Compositions and methods comprising a lipolytic enzyme variant

ABSTRACT

The present invention provides lipolytic enzyme variants. Specifically, the present invention provides lipolytic enzyme variants having two, three, or more modifications as compared to a parent lipolytic enzyme and having at least one improved property. In addition, the present invention provides compositions comprising a lipolytic enzyme variant of the invention. The present invention also provides methods of cleaning using compositions comprising a lipolytic enzyme variant of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/906,225, filed Jan. 19, 2016, which is a 371 of InternationalApplication No. PCT/US14/47174, filed Jul. 18, 2014, and claims thebenefit of priority from U.S. provisional application U.S. Ser. No.61/856,524, filed Jul. 19, 2013, all of which are incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20171023_NB40409USCNT_SeqLst.txt created on Oct. 23, 2017 and having asize of 7 kilobytes and is filed concurrently with the specification.The sequence listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Lipolytic enzymes, including lipases and cutinases, have been employedin detergent cleaning compositions for the removal of oily stains. Onemechanism by which lipolytic enzymes function is by hydrolyzingtriglycerides to generate fatty acids. However, these enzymes are ofteninhibited by surfactants and other components present in cleaningcomposition, interfering with their ability to remove oily stains.Accordingly, the need exists for lipolytic enzymes that have improvedfunction and can be effective in the harsh environment of cleaningcompositions.

SUMMARY OF THE INVENTION

The present invention provides improved lipolytic enzymes, especiallyenzymes useful for detergent compositions. Specifically, the presentinvention provides lipolytic enzyme variants having two or moremodifications, such as a substitution, as compared to a parent lipolyticenzyme that have improved lipolytic activity, such as improvedhydrolysis of p-nitrophenyl butyrate, p-nitrophenyl caprylate, orp-nitrophenyl palmitate, increased pNPP/pNPB or pNPO/pNPB specificactivity ratio, increased pNPB/pNPP specific activity ratio, increasedthermostability, increased detergent stability, increased LAS stability,increased fabric adhesion, decreased fabric adhesion, or increasedcleaning performance. This improved activity can improve effectivenessof the variant enzyme in a wash cycle. The present invention providesvariant lipolytic enzymes, including, but not limited to, variant lipaselipolytic enzymes, that are particularly well suited to and useful in avariety of cleaning applications. The invention also provides methods ofcleaning using lipolytic enzyme variants of the present invention.

In some embodiments, the invention is a lipolytic enzyme variant or anactive fragment thereof comprising at least two, three, four, five, six,seven, eight, nine or ten or more amino acid modifications to a parentlipolytic enzyme, wherein a first amino acid modification is at aposition of the lipolytic enzyme variant selected from the groupconsisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73,75, 77, 90, 91, 94, 111, 130, 137, 154, 156, 163, 187, 189, 227, 233,252, 256, and 264, wherein the amino acid positions of the variant arenumbered by correspondence with the amino acid sequence of Thermomyceslanuginosa lipase set forth in SEQ ID NO: 1.

In some embodiments, the invention is a lipolytic enzyme variant oractive fragment thereof, wherein the variant or active fragment thereofcomprises amino acid modifications of Table 2-2, wherein the amino acidpositions of the variant are numbered by correspondence with the aminoacid sequence of Thermomyces lanuginosa lipase set forth in SEQ ID NO:1.

In some of the above embodiments, the variant or active fragment haslipolytic activity. In some of the above embodiments, the variant oractive fragment has a performance index (pI) relative to the parentlipolytic enzyme for hydrolysis of p-nitrophenyl butyrate, p-nitrophenylcaprylate, or p-nitrophenyl palmitate is greater than 1.0, with someinstances wherein the performance index is measured using thep-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenylpalmitate assay of Example 1.

In some of the above embodiments, the variant or active fragment hasincreased pNPP/pNPB or pNPO/pNPB specific activity ratio, increasedpNPB/pNPP specific activity ratio, increased thermostability, increaseddetergent stability, increased LAS stability, increased fabric adhesion,decreased fabric adhesion, or increased cleaning performance.

In some embodiments, the invention is a composition comprising at leastone lipolytic enzyme variant as listed above. The composition can be acleaning composition or cleaning additive. In some embodiments, theinvention further includes an additional enzyme from the groupconsisting of protease, hemicellulase, cellulase, peroxidase, lipolyticenzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase,esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase,keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase,pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase,hyaluronidase, chondroitinase, laccase, and amylase.

In one embodiment, the invention is a method hydrolyzing a fatty acidester or triglyceride comprising contacting the fatty acid ester ortriglyceride with a lipolytic enzyme variant listed above. In oneembodiment, the invention is a method of cleaning, comprising contactinga surface or an item with a cleaning composition comprising at least onelipolytic enzyme variant listed above.

DESCRIPTION OF THE INVENTION

The present invention provides improved lipolytic enzymes, especiallyenzymes useful for detergent compositions. Specifically, the presentinvention provides lipolytic enzyme variants having two or moremodifications, such as a substitution, as compared to a parent lipolyticenzyme that have improved lipolytic activity, such as improvedhydrolysis of fatty acid esters or triglycerides, or for example,p-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenylpalmitate. The present invention provides variant lipolytic enzymes,including, but not limited to, variant lipase lipolytic enzymes, thatare particularly well suited to and useful in a variety of cleaningapplications. The invention includes compositions comprising at leastone of the variant lipolytic enzymes (e.g., variant lipases) set forthherein. Some such compositions comprise detergent compositions. Theinvention provides Thermomyces species variant lipolytic enzymes andcompositions comprising one or more such variant lipases. The lipolyticenzyme variants of the present invention can be combined with otherenzymes useful in detergent compositions. The invention also providesmethods of cleaning using lipolytic enzyme variants of the presentinvention.

The invention includes enzyme variants of lipolytic enzymes having twoor more modifications from a parent lipolytic enzyme. A parent lipolyticenzyme can be the wild-type enzyme or any starting reference lipolyticenzyme from which the variant lipolytic enzyme was derived.

Additionally, the invention provides modifications, such as asubstitution, at two, three, or more amino acid positions in a lipolyticenzyme which can be useful in a detergent composition where favorablemodifications result in an improved performing index (pI) for lipolyticactivity compared to a parent lipolytic enzyme, such as SEQ ID NO:1, orto a reference lipase enzyme, such as SEQ ID NO: 2. These amino acidpositions can be considered useful positions for combinatorialmodifications to a parent lipolytic enzyme.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although many methodsand materials similar or equivalent to those described herein find usein the practice of the present invention, some methods and materials aredescribed herein. Accordingly, the terms defined immediately below aremore fully described by reference to the specification as a whole. Allpatents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference.

Also, as used herein, the singular terms “a,” “an,” and “the” includethe plural reference unless the context clearly indicates otherwise.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. It is to be understood thatthis invention is not limited to the particular methodology, protocols,and reagents described, as these may vary, depending upon the context inwhich they are used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification include every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

A “protein” or “polypeptide” comprises a polymeric sequence of aminoacid residues. The terms “protein” and “polypeptide” are usedinterchangeably herein. The single and 3-letter code for amino acids asdefined in conformity with the IUPAC-IUB Joint Commission on BiochemicalNomenclature (JCBN) is used through out this disclosure. It is alsounderstood that a polypeptide can be coded for by more than onenucleotide sequence due to the degeneracy of the genetic code. Mutationscan be named by the one letter code for the parent amino acid, followedby a number and then the one letter code for the variant amino acid. Forexample, mutating glycine (G) at position 87 to serine (S) can berepresented as “G087S” or “G87S”. Multiple mutations can be indicated byinserting a “−,” “+,” or “,” between the mutations. For example,mutations at positions 87 and 90 can be represented as either“G087S-A090Y” or “G87S−A90Y” or “G87S+A90Y” or “G087S+A090Y”.

The terms “derived from” and “obtained from” refer not only to alipolytic enzyme produced or producible by a strain of the organism inquestion, but also a lipolytic enzyme encoded by a DNA sequence isolatedfrom such strain and produced in a host organism containing such DNAsequence. Additionally, the term refers to a lipolytic enzyme which isencoded by a DNA sequence of synthetic and/or cDNA origin and which hasthe identifying characteristics of the lipolytic enzyme in question. Toexemplify, “lipolytic enzymes derived from Thermomyces lanuginosa”refers to those enzymes having lipolytic activity which are naturallyproduced by Thermomyces lanuginosa, as well as to lipolytic enzymes likethose produced by Thermomyces lanuginosa sources but which through theuse of genetic engineering techniques are produced by non-Thermomyceslanuginosa organisms transformed with a nucleic acid encoding thelipolytic enzymes.

As used herein, “% identity or percent identity” refers to sequencesimilarity. Percent identity may be determined using standard techniquesknown in the art (See e.g., Smith and Waterman, Adv. Appl. Math. 2:482[1981]; Needleman and Wunsch, J. Mol. Biol. 48:443 [1970]; Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; software programssuch as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package (Genetics Computer Group, Madison, Wis.); and Devereuxet al., Nucl. Acid Res. 12:387-395 [1984]). One example of a usefulalgorithm is PILEUP. PILEUP creates a multiple sequence alignment from agroup of related sequences using progressive, pair-wise alignments. Itcan also plot a tree showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (See, Feng and Doolittle, J. Mol. Evol.35:351-360 [1987]). The method is similar to that described by Higginsand Sharp (See, Higgins and Sharp, CABIOS 5:151-153 [1989]). UsefulPILEUP parameters include a default gap weight of 3.00, a default gaplength weight of 0.10, and weighted end gaps. Other useful algorithm isthe BLAST algorithms described by Altschul et al., (See, Altschul etal., J. Mol. Biol. 215:403-410 [1990]; and Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787 [1993]). The BLAST program usesseveral search parameters, most of which are set to the default values.

The NCBI BLAST algorithm finds the most relevant sequences in terms ofbiological similarity but is not recommended for query sequences of lessthan 20 residues (Altschul, S F et al. (1997) Nucleic Acids Res.25:3389-3402 and Schaffer, A A et al. (2001) Nucleic Acids Res.29:2994-3005). Example default BLAST parameters for a nucleic acidsequence searches are:

Neighboring words threshold: 11 E-value cutoff: 10 Scoring Matrix:NUC.3.1 (match = 1, mismatch = −3) Gap Opening:  5 Gap Extension:  2and the following parameters for amino acid sequence searches:

Word size:  3 E-value cutoff: 10 Scoring Matrix: BLOSUM62 Gap Opening:11 Gap extension:  1

A percent (%) amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “reference” sequence including any gaps created by theprogram for optimal/maximum alignment. If a sequence is 90% identical toSEQ ID NO: A, SEQ ID NO: A is the “reference” sequence. BLAST algorithmsrefer the “reference” sequence as “query” sequence.

The CLUSTAL W algorithm is another example of a sequence alignmentalgorithm. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680.Default parameters for the CLUSTAL W algorithm are:

Gap opening penalty: 10.0 Gap extension penalty:  0.05 Protein weightmatrix: BLOSUM series DNA weight matrix: IUB Delay divergent sequences%: 40 Gap separation distance:  8 DNA transitions weight:  0.50 Listhydrophilic residues: GPSNDQEKR Use negative matrix: OFF Toggle Residuespecific penalties: ON Toggle hydrophilic penalties: ON Toggle end gapseparation penalty OFF.

In CLUSTAL algorithms, deletions occurring at either terminus areincluded. For example, a variant with five amino acid deletion at eitherterminus (or within the polypeptide) of a polypeptide of 500 amino acidswould have a percent sequence identity of 99% (495/500 identicalresidues×100) relative to the “reference” polypeptide. Such a variantwould be encompassed by a variant having “at least 99% sequenceidentity” to the polypeptide.

A polypeptide of interest may be said to be “substantially identical” toa reference polypeptide if the polypeptide of interest comprises anamino acid sequence having at least about 60%, least about 65%, leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or at leastabout 99.5% sequence identity to the amino acid sequence of thereference polypeptide. The percent identity between two suchpolypeptides can be determined manually by inspection of the twooptimally aligned polypeptide sequences or by using software programs oralgorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. Oneindication that two polypeptides are substantially identical is that thefirst polypeptide is immunologically cross-reactive with the secondpolypeptide. Typically, polypeptides that differ by conservative aminoacid substitutions are immunologically cross-reactive. Thus, apolypeptide is substantially identical to a second polypeptide, forexample, where the two peptides differ only by a conservative amino acidsubstitution or one or more conservative amino acid substitutions.

A nucleic acid of interest may be said to be “substantially identical”to a reference nucleic acid if the nucleic acid of interest comprises anucleotide sequence having least about 60%, least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or at leastabout 99.5% sequence identity to the nucleotide sequence of thereference nucleic acid. The percent identity between two such nucleicacids can be determined manually by inspection of the two optimallyaligned nucleic acid sequences or by using software programs oralgorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. Oneindication that two nucleic acid sequences are substantially identicalis that the two nucleic acid molecules hybridize to each other understringent conditions (e.g., within a range of medium to highstringency).

A nucleic acid or polynucleotide is “isolated” when it is partially orcompletely separated from other components, including but not limited tofor example, other proteins, nucleic acids, cells, etc. Similarly, apolypeptide, protein or peptide is “isolated” when it is partially orcompletely separated from other components, including but not limited tofor example, other proteins, nucleic acids, cells, etc. On a molarbasis, an isolated species is more abundant than are other species in acomposition. For example, an isolated species may comprise at leastabout 50%, about 70%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% (on a molar basis) of all macromolecularspecies present. Preferably, the species of interest is purified toessential homogeneity (i.e., contaminant species cannot be detected inthe composition by conventional detection methods). Purity andhomogeneity can be determined using a number of techniques well known inthe art, such as agarose or polyacrylamide gel electrophoresis of aprotein or nucleic acid sample, followed by visualization upon staining.If desired, a high-resolution technique, such as high performance liquidchromatography (HPLC) or a similar means can be utilized forpurification of the material.

The term “purified” as applied to nucleic acids or polypeptidesgenerally denotes a nucleic acid or polypeptide that is essentially freefrom other components as determined by analytical techniques well knownin the art (e.g., a purified polypeptide or polynucleotide forms adiscrete band in an electrophoretic gel, chromatographic eluate, and/ora media subjected to density gradient centrifugation). For example, anucleic acid or polypeptide that gives rise to essentially one band inan electrophoretic gel is “purified.” A purified nucleic acid orpolypeptide is at least about 50% pure, usually at least about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent byweight on a molar basis). In a related sense, the invention providesmethods of enriching compositions for one or more molecules of theinvention, such as one or more polypeptides or polynucleotides of theinvention. A composition is enriched for a molecule when there is asubstantial increase in the concentration of the molecule afterapplication of a purification or enrichment technique. A substantiallypure polypeptide or polynucleotide of the invention (e.g., substantiallypure variant lipolytic enzyme or polynucleotide encoding a variantlipolytic enzyme of the invention, respectively) will typically compriseat least about 55%, about 60%, about 70%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98, about 99%, about 99.5% or more by weight (on amolar basis) of all macromolecular species in a particular composition.

The position of an amino acid residue in a given amino acid sequence istypically numbered herein using the numbering of the position of thecorresponding amino acid residue of the Thermomyces lanuginosa lipaseTLL amino acid sequence shown in SEQ ID NO: 1. The T. lanuginosa lipaseTLL amino acid sequence of SEQ ID NO: 1, thus serves as a referenceparent sequence. A given amino acid sequence, such as a variantlipolytic enzyme amino acid sequence described herein, can be alignedwith the TLL sequence (SEQ ID NO: 1) using an alignment algorithm asdescribed herein, and an amino acid residue in the given amino acidsequence that aligns (preferably optimally aligns) with an amino acidresidue in the TLL sequence can be conveniently numbered by reference tothe corresponding amino acid residue in the lipase TLL sequence.

Lipolytic Enzymes of the Invention

As used herein, a lipolytic enzyme includes an enzyme, polypeptide, orprotein exhibiting a lipid degrading capability such as a capability ofdegrading a triglyceride or a phospholipid. The lipolytic enzyme can be,for example, a lipase, a phospholipase, an esterase, a polyesterase, ora cutinase. Lipolytic enzymes can be lipolytic enzymes having analpha/beta hydrolase fold. These enzymes typically have a catalytictriad of serine, aspartic acid and histidine residues. The alpha/betahydrolases include lipases and cutinases. Cutinases show little, if any,interfacial activation, where lipases often undergo a conformationalchange in the presence of a lipid-water interface (Longhi and Cambillau(1999) Biochimica et Biophysica Acta 1441:185-96). An active fragment ofa lipolytic enzyme is a portion of a lipolytic enzyme that retains alipid degrading capability. An active fragment retains the catalytictriad. As used herein, lipolytic activity can be determined according toany procedure known in the art (see, e.g., Gupta et al., Biotechnol.Appl. Biochem., 37:63-71, 2003; U.S. Pat. No. 5,990,069; andInternational Patent Publication No. WO 96/18729A1).

In some embodiments, lipolytic enzymes of the present invention are □/□hydrolases. In some embodiments, lipolytic enzymes of the presentinvention are lipases. In some embodiments, lipolytic enzymes of thepresent invention are cutinases. In some embodiments, lipolytic enzymesof the present invention are polyesterases.

Productive Positions of Lipolytic Enzymes

The invention provides modifications, such as a substitution, at two,three, four, five, six, seven, eight, nine or ten or more amino acidpositions in a lipolytic enzyme which can be useful in a detergentcomposition where favorable modifications result in an improvedperforming index (pI) for lipolytic activity, such as improvedhydrolysis of fatty acid esters or triglycerides, or for example,p-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenylpalmitate, increased pNPP/pNPB or pNPO/pNPB specific activity ratio,increased pNPB/pNPP specific activity ratio, increased thermostability,increased detergent stability, increased LAS stability, increased fabricadhesion, decreased fabric adhesion, or increased cleaning performance,compared to the parent lipolytic enzyme. These amino acid positions canbe considered useful positions for combinatorial modifications to aparent lipolytic enzyme.

Lipolytic enzyme amino acid positions found to be useful positions canhave different modifications that are suitable for use in a detergentcomposition. Modifications can include an insertion, deletion orsubstitution at the particular position. In one embodiment, amodification is a substitution.

In some embodiments, the invention is a lipolytic enzyme variant or anactive fragment thereof comprising at least two, three, four, five, six,seven, eight, nine or ten or more amino acid modifications to a parentlipolytic enzyme, wherein a first amino acid modification is at aposition of the lipolytic enzyme variant selected from the groupconsisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73,75, 77, 90, 91, 94, 111, 130, 137, 154, 156, 163, 187, 189, 227, 233,252, 256, and 264, wherein the amino acid positions of the variant arenumbered by correspondence with the amino acid sequence of Thermomyceslanuginosa lipase set forth in SEQ ID NO: 1. In some embodiments, theinvention is a lipolytic enzyme variant or an active fragment thereof,wherein the at least two, three, four, five, six, seven, eight, nine orten or more amino acid modifications to a parent lipolytic enzyme are ata position of the lipolytic enzyme variant selected from the groupconsisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73,75, 77, 90, 91, 94, 111, 130, 137, 154, 156, 163, 187, 189, 227, 233,252, 256, and 264, wherein the amino acid positions of the variant arenumbered by correspondence with the amino acid sequence of Thermomyceslanuginosa lipase set forth in SEQ ID NO: 1.

In some embodiments, a lipolytic enzyme variant or active fragmentthereof of the invention can have two or more modifications, where themodifications are A018K/D027S, A018K/E045F, A018K/L075D, A018K/L075Q,A018K/T189D, D027E/G163P, D027N/E056K, D027N/N233Q, D027S/N033D,D027S/P256T, D111A/L264R, D130A/L264R, D130A/T189Q, D130A/V187N,D130A/V187T, E045F/N073R, E045F/T189D, F051T/L075R, G023K/D130A,G023K/L264R, G023Q/L075Q, K024A/L075Q, K024A/V154I, L075Q/D130A,L075Q/G156W, L075R/L264R, L227M/L264R, N011K/D027S, N033D/E045F,N073R/L075D, N094R/G156W, P029E/N033D, S058M/L264R, V187T/L264R,A018K/A049V/L075Q, A018K/A049V/V187T, A018K/D027S/E045F,A018K/D027S/N073R, A018K/D027S/T189D, A018K/E045F/N073R,A018K/E045F/T189D, A018K/E045F/V187T, A018K/G023K/G156W,A018K/G023K/L075Q, A018K/G023Q/L075Q, A018K/K024A/V154I,A018K/L075D/T189D, A018K/L075Q/G156W, A018K/L075Q/V187T,A018K/N033D/L075D, A018K/N033D/T189D, A018K/N073R/L075D,A018K/P029E/T189D, A018K/V154I/G156W, A049V/L075Q/T189D,A049V/V187T/T189D, D027E/S058M/G163P, D027N/N233Q/P256T,D027N/S058M/P256T, D027Q/N233Q/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/N073R/L075D,D027S/N233Q/P256T, D027S/S058M/P256T, D111A/D130A/L264R,D130A/T189Q/L264R, D130A/V154I/G156W, D130A/V187N/L264R,D130A/V187T/L264R, E045F/L075D/T189D, E045F/N073R/L075D,E056K/D130A/L264R, E056K/D130A/T189Q, F051T/I252Q/L264R,G023K/D130A/L264R, G023K/D130A/V187T, G023K/E056K/V187T,G023K/L075R/L264R, G023K/V187N/L264R, G023K/V187T/L264R,G023Q/A049V/T189D, G023Q/D111A/L264R, G023Q/D130A/G156W,G023Q/E045F/V077I, G023Q/E056K/L075R, G023Q/K024A/G156W,G023Q/L075Q/V077I, G023Q/L075Q/V187T, G163P/L227M/L264R,I090F/N233Q/P256T, K024A/D130A/V154I, K024A/L075Q/G156W,K024A/L075Q/V077I, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/D130A/V187T, L075Q/G156W/T189D, L075Q/G156W/V187N,L075Q/G156W/V187T, L075Q/N094R/V154I, L075Q/V077I/T189D,L075Q/V154I/V187T, L075Q/V187N/L264R, L075Q/V187T/L264R,L075Q/V187T/T189D, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187N/L264R, L075R/V187T/L264R, N011K/D027S/S058M,N011K/N233Q/P256T, N033D/E045F/L075D, N033D/E045F/N073R,N033D/N073R/T189D, N073R/L075D/T189D, N094R/D130A/V187T,P029E/E045F/L075D, P029E/L075D/T189D, P029E/N033D/E045F,P029E/N033D/L075D, P029E/N073R/L075D, Q004D/D027S/P256T,Q004D/N233Q/P256T, Q004D/S058M/N233Q, S058M/L227M/L264R,V077I/D130A/V154I, V077I/V187A/T189D, V187N/T189Q/L264R,A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/N073R, A018K/D027S/E045F/T189D,A018K/D027S/L075D/T189D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/E045F/A049V/G156W,A018K/E045F/A049V/V187T, A018K/E045F/L075D/T189D,A018K/E045F/L075Q/T189D, A018K/E045F/L075Q/V187T,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/T189D,A018K/G023Q/A049V/V187T, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/G156W/V187T,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V077I,A018K/G023Q/L075Q/V187T, 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the amino acid positions of the variant are numbered bycorrespondence with the amino acid sequence of Thermomyces lanuginosalipase set forth in SEQ ID NO: 1. In some embodiments, the variant oractive fragment has lipolytic activity, for example, a performance index(pI) relative to the parent lipolytic enzyme for hydrolysis ofp-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenylpalmitate is greater than 1.0 at a pH of 6 or 8.

In any of the above embodiments, the invention provides variantlipolytic enzymes of the invention that exhibit one of more of thefollowing properties: improved lipolytic activity, such as improvedhydrolysis of p-nitrophenyl butyrate, p-nitrophenyl caprylate, orp-nitrophenyl palmitate, increased pNPP/pNPB or pNPO/pNPB specificactivity ratio, increased pNPB/pNPP specific activity ratio, increasedthermostability, increased detergent stability, increased LAS stability,increased fabric adhesion, decreased fabric adhesion, increased cleaningperformance, improved hand wash performance, improved hand or manualdishwashing performance, improved automatic dishwashing performance,improved laundry performance, and/or improved stability relative to aparent lipolytic enzyme (e.g., wild-type lipolytic enzyme, such as awild-type lipase) or reference enzyme (such as SEQ ID NO: 2).

These amino acid positions can be considered useful positions forcombinatorial modifications to a parent lipolytic enzyme. Thus, theinvention includes lipolytic enzymes having one or more modifications atany of the above positions.

Polypeptides of the Invention

The present invention provides novel polypeptides, which may becollectively referred to as “polypeptides of the invention.”Polypeptides of the invention include isolated, recombinant,substantially pure, or non-naturally occurring variant lipolytic enzymepolypeptides, including for example, variant lipolytic enzymepolypeptides, having enzymatic activity (e.g., lipolytic activity). Insome embodiments, polypeptides of the invention are useful in cleaningapplications and can be incorporated into cleaning compositions that areuseful in methods of cleaning an item or a surface (e.g., of surface ofan item) in need of cleaning.

In some embodiments, the lipolytic enzyme variant can be a variant of aparent lipolytic enzyme from the Genus Thermomyces. Various lipolyticenzymes have been found in the genus Thermomyces that have a highidentity to each other and to the lipolytic enzyme from Thermomyceslanuginosa (TLL) as shown in SEQ ID NO: 1. In other embodiments, thelipolytic enzyme variant can be a variant of a parent lipolytic enzymesuch as Verrucosispora, Saccharomonospora, Streptomyces, Micromonospora,Streptosporangium, Amycolatopsis, Cellulomonas, Actinosynnema,Kribbella, Thermomonospora, Deinococcus, Kineococcus, Nocardiopsis,Frankia, Jonesia, Pseudomonas, Acidovorax or Nocardioidaceae.

The sequence of the lipolytic enzyme from Thermomyces lanuginosa (TLL)is shown below:

(SEQ ID NO: 1) EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQP NIPDIPAHLWYFGLIGTCL.

In some embodiments, the lipolytic enzyme variant can be a varianthaving 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identityto a lipolytic enzyme from the genus Thermomyces. In some embodiments,the lipolytic enzyme variant can be a variant having 50, 60, 70, 75, 80,85, 90, 95, 96, 97, 98, 99 or 100% identity to a lipolytic enzyme fromthe species Thermomyces lanuginosa, the lipolytic enzyme having thesequence shown in SEQ ID NO: 1.

In a particular embodiment, the invention is an enzyme derived from thegenus Thermomyces. In a particular embodiment, the invention is anenzyme derived from a lipolytic enzyme from the species Thermomyceslanuginosa, the lipolytic enzyme having the sequence shown in SEQ ID NO:1.

Described are compositions and methods relating to lipase cloned fromThermomyces lanuginosa (TLL). The compositions and methods are based, inpart, on the observation that cloned and expressed TLL has carboxylicester hydrolase activity (acts on carboxylic acid esters) in thepresence of a detergent compositions. TLL also demonstrates excellentstability in detergent compositions, even in the presence of proteaseenzyme. These features of TLL makes it well suited for use in a varietyof cleaning applications, where the enzyme can hydrolyze lipids in thepresence of surfactants and other components found in detergentcompositions.

While TLL shows activity against a variety of natural and syntheticsubstrates, the enzyme has shown a preference for C4-C16 substrates.This specificity makes TLL well suited for hydrolysis of short-chaintriglycerides and for performing transesterification reactions involvingshort-chain fatty acids.

In any of the above embodiments, the variant lipolytic enzyme of theinvention can have improved lipolytic activity on C4-C16 substratesrelative to the parent lipolytic enzyme. In some embodiments, variantsof the invention have an increased pNPP/pNPB or pNPO/pNPB specificactivity ratio. In some embodiments, variants of the invention have anincreased pNPB/pNPP specific activity ratio. In any of the aboveembodiments, the variant lipolytic enzyme of the invention can have aperformance index (pI) relative to a parent lipolytic enzyme (e.g. SEQID NO:1) for hydrolysis of p-nitrophenyl butyrate, p-nitrophenylcaprylate, or p-nitrophenyl palmitate is greater than 1.0. This can bemeasured at pH 6 or 8. In any of the above embodiments, the variantlipolytic enzyme of the invention can have a performance index (pI)relative to a parent lipolytic enzyme (e.g. SEQ ID NO:1) for detergentstability that is greater than 1.0. In any of the above embodiments, thevariant lipolytic enzyme of the invention can have a performance index(pI) relative to a parent lipolytic enzyme (e.g. SEQ ID NO:1) for LASstability that is greater than 1.0. In any of the above embodiments, thevariant lipolytic enzyme of the invention can have a performance index(pI) relative to a parent lipolytic enzyme (e.g. SEQ ID NO:1) for fabricadhesion that is greater than 1.0. In any of the above embodiments, thevariant lipolytic enzyme of the invention can have a performance index(pI) relative to a parent lipolytic enzyme (e.g. SEQ ID NO:1) for fabricadhesion that is less than 1.0. In any of the above embodiments, thevariant lipolytic enzyme of the invention can have a performance index(pI) relative to a parent lipolytic enzyme (e.g. SEQ ID NO: 1) forcleaning performance that is greater than 1.0. This can be measuredusing a CS-61 microswatch assay using Tide® half dose+adjuvant or Tide®full dose. In any of the above embodiments, the variant lipolytic enzymeof the invention can have a performance index (pI) relative to areference lipolytic enzyme (e.g. SEQ ID NO: 2) that is greater than 1.0for hydrolysis of p-nitrophenyl butyrate, p-nitrophenyl caprylate, orp-nitrophenyl palmitate, detergent stability, LAS stability, fabricadhesion, or cleaning performance. In any of the above embodiments, thevariant lipolytic enzyme of the invention can have a performance index(pI) relative to a reference lipolytic enzyme (e.g. SEQ ID NO: 2) thatis less than 1.0 for fabric adhesion.

The sequence of the reference lipolytic enzyme, which is a variant fromThermomyces lanuginosa (TLL) is shown below:

(SEQ ID NO: 2) EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVRRRDIVKIEGIDATGGNNQP NIPDIPAHLWYFGLIGTCL.

In several of the above embodiments, the present compositions andmethods provide a variant TLL polypeptide. The parent TLL polypeptideThermomyces lanuginosa lipase was described in WO1994025577. The matureTLL polypeptide has the amino acid sequence of SEQ ID NO: 1. Similar,substantially identical TLL polypeptides may occur in nature, e.g., inother strains or isolates of T. lanuginosa. These and other recombinantTLL polypeptides are encompassed by the present compositions andmethods.

In any of the above embodiments, the invention includes an isolated,recombinant, substantially pure, or non-naturally occurring variantlipolytic enzyme having lipolytic activity, which polypeptide comprisesa polypeptide sequence having at least about 85%, at least about 86%, atleast about 87%, at least about 88%, at least about 89%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, at least about 99.5%, or100% sequence identity to a parent lipolytic enzyme as provided herein.

In some embodiments, the variant polypeptide is a variant having aspecified degree of amino acid sequence homology to the exemplified TLLpolypeptide, e.g., at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or even atleast 99% sequence homology to the amino acid sequence of SEQ ID NO: 1.Homology can be determined by amino acid sequence alignment, e.g., usinga program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is an isolated, recombinant, substantially pure, ornon-naturally occurring sequence which encodes a variant lipolyticenzyme having lipolytic activity, said variant lipolytic enzyme (e.g.,variant lipase) comprising an amino acid sequence which differs from theamino acid sequence of SEQ ID NO: 1 by no more than 50, no more than 40,no more than 30, no more than 35, no more than 25, no more than 20, nomore than 19, no more than 18, no more than 17, no more than 16, no morethan 15, no more than 14, no more than 13, no more than 12, no more than11, no more than 10, no more than 9, no more than 8, no more than 7, nomore than 6, no more than 5, no more than 4, no more than 3, or no morethan 2 amino acid residue(s), wherein amino acid positions of thevariant lipase are numbered according to the numbering of correspondingamino acid positions in the amino acid sequence of Thermomyceslanuginosa lipase TLL shown in SEQ ID NO: 1 as determined by alignmentof the variant lipolytic enzyme amino acid sequence with the Thermomyceslanuginosa lipase TLL amino acid sequence.

In some embodiments, the present invention relates to isolatedpolypeptides having lipase activity that are encoded by polynucleotidesthat hybridize under preferably very low stringency conditions, morepreferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with the mature polypeptide coding sequence of SEQID NO: 1, or a full-length complementary strand thereof (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, New York).

As noted above, the variant lipolytic enzyme polypeptides of theinvention have enzymatic activities (e.g., lipolytic activities) andthus are useful in cleaning applications, including but not limited to,methods for cleaning dishware items, tableware items, fabrics, and itemshaving hard surfaces (e.g., the hard surface of a table, table top,wall, furniture item, floor, ceiling, etc.). Exemplary cleaningcompositions comprising one or more variant lipolytic enzymepolypeptides of the invention are described infra. The enzymaticactivity (e.g., lipolytic enzyme activity) of a variant lipolytic enzymepolypeptide of the invention can be determined readily using procedureswell known to those of ordinary skill in the art. The performance ofvariant lipolytic enzymes of the invention in removing stains (e.g., alipid stain), cleaning hard surfaces, or cleaning laundry, dishware ortableware item(s) can be readily determined using procedures well knownin the art.

A polypeptide of the invention can be subject to various changes, suchas one or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where such changes donot substantially alter the enzymatic activity of the polypeptide.Similarly, a nucleic acid of the invention can also be subject tovarious changes, such as one or more substitutions of one or morenucleic acids in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (e.g., mutation in a nucleotide sequence results in a silentmutation in the amino acid sequence, for example when the encoded aminoacid is not altered by the nucleic acid mutation) or non-silentvariation, one or more deletions of one or more nucleic acids (orcodons) in the sequence, one or more additions or insertions of one ormore nucleic acids (or codons) in the sequence, and/or cleavage of orone or more truncations of one or more nucleic acids (or codons) in thesequence. Many such changes in the nucleic acid sequence may notsubstantially alter the enzymatic activity of the resulting encodedvariant lipolytic enzyme compared to the variant lipolytic enzymeencoded by the original nucleic acid sequence. A nucleic acid of theinvention can also be modified to include one or more codons thatprovide for optimum expression in an expression system (e.g., bacterialexpression system), while, if desired, said one or more codons stillencode the same amino acid(s).

In some embodiments, the present invention provides a genus ofpolypeptides comprising variant lipolytic enzyme polypeptides having thedesired enzymatic activity (e.g., lipolytic enzyme activity or cleaningperformance activity) which comprise sequences having the amino acidsubstitutions described herein and also which comprise one or moreadditional amino acid substitutions, such as conservative andnon-conservative substitutions, wherein the polypeptide exhibits,maintains, or approximately maintains the desired enzymatic activity(e.g., lipolytic enzyme activity or lipase activity, as reflected in thecleaning activity or performance of the variant lipolytic enzyme). Aminoacid substitutions in accordance with the invention may include, but arenot limited to, one or more non-conservative substitutions and/or one ormore conservative amino acid substitutions. A conservative amino acidresidue substitution typically involves exchanging a member within onefunctional class of amino acid residues for a residue that belongs tothe same functional class (identical amino acid residues are consideredfunctionally homologous or conserved in calculating percent functionalhomology). A conservative amino acid substitution typically involves thesubstitution of an amino acid in an amino acid sequence with afunctionally similar amino acid. For example, alanine, glycine, serine,and threonine are functionally similar and thus may serve asconservative amino acid substitutions for one another. Aspartic acid andglutamic acid may serve as conservative substitutions for one another.Asparagine and glutamine may serve as conservative substitutions for oneanother. Arginine, lysine, and histidine may serve as conservativesubstitutions for one another. Isoleucine, leucine, methionine, andvaline may serve as conservative substitutions for one another.Phenylalanine, tyrosine, and tryptophan may serve as conservativesubstitutions for one another.

Other conservative amino acid substitution groups can be envisioned. Forexample, amino acids can be grouped by similar function or chemicalstructure or composition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For instance, an aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E); non-polar uncharged residues,Cysteine (C), Methionine (M), and Proline (P); hydrophilic unchargedresidues: Serine (S), Threonine (T), Asparagine (N), and Glutamine (Q).Additional groupings of amino acids are well-known to those of skill inthe art and described in various standard textbooks. Listing of apolypeptide sequence herein, in conjunction with the above substitutiongroups, provides an express listing of all conservatively substitutedpolypeptide sequences.

More conservative substitutions exist within the amino acid residueclasses described above, which also or alternatively can be suitable.Conservation groups for substitutions that are more conservativeinclude: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, and asparagine-glutamine. Thus, forexample, in some embodiments, the invention provides an isolated orrecombinant variant lipolytic enzyme polypeptide (e.g., variant lipase)having lipolytic activity, said variant lipolytic enzyme polypeptidecomprising an amino acid sequence having at least about 90%, about 95%,about 96%, about 97%, about 98%, about 99%, or about 99.5% sequenceidentity to the amino acid sequence of SEQ ID NO: 1. A conservativesubstitution of one amino acid for another in a variant lipolytic enzymeof the invention is not expected to alter significantly the enzymaticactivity or cleaning performance activity of the variant lipolyticenzyme. Enzymatic activity or cleaning performance activity of theresultant lipolytic enzyme can be readily determined using the standardassays and the assays described herein.

Conservatively substituted variations of a polypeptide sequence of theinvention (e.g., variant lipolytic enzymes of the invention) includesubstitutions of a small percentage, sometimes less than about 25%,about 20%, about 15%, about 14%, about 13%, about 12%, about 11%, about10%, about 9%, about 8%, about 7%, or about 6% of the amino acids of thepolypeptide sequence, or less than about 5%, about 4%, about 3%, about2%, or about 1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup.

Nucleic Acids of the Invention

The invention provides isolated, non-naturally occurring, or recombinantnucleic acids (also referred to herein as “polynucleotides”), which maybe collectively referred to as “nucleic acids of the invention” or“polynucleotides of the invention”, which encode polypeptides of theinvention. Nucleic acids of the invention, including all describedbelow, are useful in recombinant production (e.g., expression) ofpolypeptides of the invention, typically through expression of a plasmidexpression vector comprising a sequence encoding the polypeptide ofinterest or fragment thereof. As discussed above, polypeptides includevariant lipolytic enzyme polypeptides, including variant lipasepolypeptides having enzymatic activity (e.g., lipolytic activity) whichare useful in cleaning applications and cleaning compositions forcleaning an item or a surface (e.g., surface of an item) in need ofcleaning.

In some embodiments, the invention provides an isolated, recombinant,substantially pure, or non-naturally occurring nucleic acid comprising anucleotide sequence encoding any polypeptide (including any fusionprotein, etc.) of the invention described above in the section entitled“Polypeptides of the Invention” and elsewhere herein. The invention alsoprovides an isolated, recombinant, substantially pure, ornon-naturally-occurring nucleic acid comprising a nucleotide sequenceencoding a combination of two or more of any polypeptides of theinvention described above and elsewhere herein.

Nucleic acids of the invention can be generated by using any suitablesynthesis, manipulation, and/or isolation techniques, or combinationsthereof. For example, a polynucleotide of the invention may be producedusing standard nucleic acid synthesis techniques, such as solid-phasesynthesis techniques that are well-known to those skilled in the art.The synthesis of the nucleic acids of the invention can be alsofacilitated (or alternatively accomplished) by any suitable method knownin the art, including but not limited to chemical synthesis using theclassical phosphoramidite method (See e.g., Beaucage et al. TetrahedronLetters 22:1859-69 (1981)); or the method described by Matthes et al.(See, Matthes et al., EMBO J. 3:801-805 (1984), as is typicallypracticed in automated synthetic methods. Nucleic acids of the inventionalso can be produced by using an automatic DNA synthesizer. Customizednucleic acids can be ordered from a variety of commercial sources (e.g.,The Midland Certified Reagent Company, the Great American Gene Company,Operon Technologies Inc., and DNA2.0). Other techniques for synthesizingnucleic acids and related principles are known in the art (See e.g.,Itakura et al., Ann. Rev. Biochem. 53:323 (1984); and Itakura et al.,Science 198:1056 (1984)).

Methods for Making Modified Variant Lipolytic Enzymes of the Invention

A variety of methods are known in the art that are suitable forgenerating modified polynucleotides of the invention that encode variantlipolytic enzymes of the invention, including, but not limited to, forexample, site-saturation mutagenesis, scanning mutagenesis, insertionalmutagenesis, deletion mutagenesis, random mutagenesis, site-directedmutagenesis, and directed-evolution, as well as various otherrecombinatorial approaches. Methods for making modified polynucleotidesand proteins (e.g., variant lipolytic enzymes) include DNA shufflingmethodologies, methods based on non-homologous recombination of genes,such as ITCHY (See, Ostermeier et al., 7:2139-44 (1999)), SCRACHY (See,Lutz et al. 98:11248-53 (2001)), SHIPREC (See, Sieber et al., 19:456-60(2001)), and NRR (See, Bittker et al., 20:1024-9 (2001); Bittker et al.,101:7011-6 (2004)), and methods that rely on the use of oligonucleotidesto insert random and targeted mutations, deletions and/or insertions(See, Ness et al., 20:1251-5 (2002); Coco et al., 20:1246-50 (2002); Zhaet al., 4:34-9 (2003); Glaser et al., 149:3903-13 (1992)).

Vectors, Cells, and Methods for Producing Variant Lipolytic Enzymes ofthe Invention

The present invention provides isolated or recombinant vectorscomprising at least one polynucleotide of the invention described herein(e.g., a polynucleotide encoding a variant lipolytic enzyme of theinvention described herein), isolated or recombinant expression vectorsor expression cassettes comprising at least one nucleic acid orpolynucleotide of the invention, isolated, substantially pure, orrecombinant DNA constructs comprising at least one nucleic acid orpolynucleotide of the invention, isolated or recombinant cellscomprising at least one polynucleotide of the invention, cell culturescomprising cells comprising at least one polynucleotide of theinvention, cell cultures comprising at least one nucleic acid orpolynucleotide of the invention, and compositions comprising one or moresuch vectors, nucleic acids, expression vectors, expression cassettes,DNA constructs, cells, cell cultures, or any combination or mixturesthereof.

In some embodiments, the invention provides recombinant cells comprisingat least one vector (e.g., expression vector or DNA construct) of theinvention which comprises at least one nucleic acid or polynucleotide ofthe invention. Some such recombinant cells are transformed ortransfected with such at least one vector. Such cells are typicallyreferred to as host cells. Some such cells comprise bacterial cells,including, but are not limited to bacillus cells, such as B. subtiliscells. Some such cells comprise fungal cells, including, but are notlimited to Trichoderma cells, such as T. reesei cells. The inventionalso provides recombinant cells (e.g., recombinant host cells)comprising at least one variant lipolytic enzyme of the invention.

In some embodiments, the invention provides a vector comprising anucleic acid or polynucleotide of the invention. In some embodiments,the vector is an expression vector or expression cassette in which apolynucleotide sequence of the invention which encodes a variantlipolytic enzyme of the invention is operably linked to one oradditional nucleic acid segments required for efficient gene expression(e.g., a promoter operably linked to the polynucleotide of the inventionwhich encodes a variant lipolytic enzyme of the invention). A vector mayinclude a transcription terminator and/or a selection gene, such as anantibiotic resistance gene that enables continuous cultural maintenanceof plasmid-infected host cells by growth in antimicrobial-containingmedia.

An expression vector may be derived from plasmid or viral DNA, or inalternative embodiments, contains elements of both. Exemplary vectorsinclude, but are not limited to pXX, pC194, pJH101, pE194, pHP13 (See,Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods forBacillus, John Wiley & Sons [1990]; suitable replicating plasmids for B.subtilis include those listed on p. 92; See also, Perego, IntegrationalVectors for Genetic Manipulations in Bacillus subtilis, in Sonenshein etal., [eds.] Bacillus subtilis and Other Gram-Positive Bacteria:Biochemistry, Physiology and Molecular Genetics, American Society forMicrobiology, Washington, D.C. [1993], pp. 615-624).

For expression and production of a protein of interest (e.g., variantlipolytic enzyme) in a cell, at least one expression vector comprisingat least one copy of a polynucleotide encoding the modified lipolyticenzyme, and preferably comprising multiple copies, is transformed intothe cell under conditions suitable for expression of the lipolyticenzyme. In some embodiments of the present invention, a polynucleotidesequence encoding the variant lipolytic enzyme (as well as othersequences included in the vector) is integrated into the genome of thehost cell, while in other embodiments, a plasmid vector comprising apolynucleotide sequence encoding the variant lipolytic enzyme remains asautonomous extra-chromosomal element within the cell. The inventionprovides both extrachromosomal nucleic acid elements as well as incomingnucleotide sequences that are integrated into the host cell genome. Thevectors described herein are useful for production of the variantlipolytic enzymes of the invention. In some embodiments, apolynucleotide construct encoding the variant lipolytic enzyme ispresent on an integrating vector that enables the integration andoptionally the amplification of the polynucleotide encoding the variantlipolytic enzyme into the bacterial chromosome. Examples of sites forintegration are well known to those skilled in the art. In someembodiments, transcription of a polynucleotide encoding a variantlipolytic enzyme of the invention is effectuated by a promoter that isthe wild-type promoter for the selected precursor lipolytic enzyme. Insome other embodiments, the promoter is heterologous to the precursorlipolytic enzyme, but is functional in the host cell. Specifically,examples of suitable promoters for use in bacterial host cells include,but are not limited to, for example, the amyE, amyQ, amyL, pstS, sacB,pSPAC, pAprE, pVeg, pHpaII promoters, the promoter of the B.stearothermophilus maltogenic amylase gene, the T. lanuginosa (BAN)amylase gene, the B. subtilis alkaline lipolytic enzyme gene, the B.clausii alkaline lipolytic enzyme gene the B. pumilis xylosidase gene,the B. thuringiensis cryIIIA, and the B. licheniformis alpha-amylasegene. Additional promoters include, but are not limited to the A4promoter, as well as phage Lambda P_(R) or P_(L) promoters, and the E.coli lac, trp or tac promoters.

Variant lipolytic enzymes of the present invention can be produced inhost cells of any suitable Gram-positive microorganism, includingbacteria and fungi. For example, in some embodiments, the variantlipolytic enzyme is produced in host cells of fungal and/or bacterialorigin. In some embodiments, the host cells are Bacillus spp.,Streptomyces spp., Escherichia spp., Aspergillus spp., Trichoderma spp.,Pseudomonas spp., Corynebacterium spp., Saccharomyces spp., or Pichiaspp. In some embodiments, the variant lipolytic enzymes are produced byBacillus sp. host cells. Examples of Bacillus sp. host cells that finduse in the production of the variant lipolytic enzymes of the inventioninclude, but are not limited to B. licheniformis, B. lentus, B.subtilis, B. amyloliquefaciens, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B.pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well asother organisms within the genus Bacillus. In some embodiments, B.subtilis host cells are used for production of variant lipolyticenzymes. U.S. Pat. Nos. 5,264,366 and 4,760,025 (RE 34,606) describevarious Bacillus host strains that can be used for producing variantlipolytic enzymes of the invention, although other suitable strains canbe used.

Several industrial bacterial strains that can be used to produce variantlipolytic enzymes of the invention include non-recombinant (i.e.,wild-type) Bacillus sp. strains, as well as variants ofnaturally-occurring strains and/or recombinant strains. In someembodiments, the host strain is a recombinant strain, wherein apolynucleotide encoding a polypeptide of interest has been introducedinto the host. In some embodiments, the host strain is a B. subtilishost strain and particularly a recombinant Bacillus subtilis hoststrain. Numerous B. subtilis strains are known, including, but notlimited to for example, 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85,B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC21332, ATCC 6051, M1113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP211strain (See e.g., Hoch et al., Genetics 73:215-228 [1973]; See also,U.S. Pat. Nos. 4,450,235 and 4,302,544, and EP 0134048, each of which isincorporated by reference in its entirety). The use of B. subtilis as anexpression host cells is well known in the art (See e.g., Palva et al.,Gene 19:81-87 [1982]; Fahnestock and Fischer, J. Bacteriol., 165:796-804[1986]; and Wang et al., Gene 69:39-47 [1988]).

In some embodiments, the Bacillus host cell is a Bacillus sp. thatincludes a mutation or deletion in at least one of the following genes,degU, degS, degR and degQ. Preferably the mutation is in a degU gene,and more preferably the mutation is degU(Hy)32 (See e.g., Msadek et al.,J. Bacteriol. 172:824-834 [1990]; and Olmos et al., Mol. Gen. Genet.253:562-567 [1997]). One suitable host strain is a Bacillus subtiliscarrying a degU32(Hy) mutation. In some embodiments, the Bacillus hostcomprises a mutation or deletion in scoC4 (See e.g., Caldwell et al., J.Bacteriol. 183:7329-7340 [2001]); spoIIE (See e.g., Arigoni et al., Mol.Microbiol. 31:1407-1415 [1999]); and/or oppA or other genes of the oppoperon (See e.g., Perego et al., Mol. Microbiol. 5:173-185 [1991]).Indeed, it is contemplated that any mutation in the opp operon thatcauses the same phenotype as a mutation in the oppA gene will find usein some embodiments of the altered Bacillus strain of the invention. Insome embodiments, these mutations occur alone, while in otherembodiments, combinations of mutations are present. In some embodiments,an altered Bacillus host cell strain that can be used to produce avariant lipolytic enzyme of the invention is a Bacillus host strain thatalready includes a mutation in one or more of the above-mentioned genes.In addition, Bacillus sp. host cells that comprise mutation(s) and/ordeletions of endogenous lipolytic enzyme genes find use. In someembodiments, the Bacillus host cell comprises a deletion of the aprE andthe nprE genes. In other embodiments, the Bacillus sp. host cellcomprises a deletion of 5 lipolytic enzyme genes, while in otherembodiments, the Bacillus sp. host cell comprises a deletion of 9lipolytic enzyme genes (See e.g., U.S. Pat. Appln. Pub. No.2005/0202535, incorporated herein by reference).

Host cells are transformed with at least one nucleic acid encoding atleast one variant lipolytic enzyme of the invention using any suitablemethod known in the art. Whether the nucleic acid is incorporated into avector or is used without the presence of plasmid DNA, it is typicallyintroduced into a microorganism, in some embodiments, preferably an E.coli cell or a competent Bacillus cell. Methods for introducing anucleic acid (e.g., DNA) into Bacillus cells or E. coli cells utilizingplasmid DNA constructs or vectors and transforming such plasmid DNAconstructs or vectors into such cells are well known. In someembodiments, the plasmids are subsequently isolated from E. coli cellsand transformed into Bacillus cells. However, it is not essential to useintervening microorganisms such as E. coli, and in some embodiments, aDNA construct or vector is directly introduced into a Bacillus host.

Those of skill in the art are well aware of suitable methods forintroducing nucleic acid or polynucleotide sequences of the inventioninto Bacillus cells (See e.g., Ferrari et al., “Genetics,” in Harwood etal. [eds.], Bacillus, Plenum Publishing Corp. [1989], pp. 57-72;Saunders et al., J. Bacteriol. 157:718-726 [1984]; Hoch et al., J.Bacteriol. 93:1925-1937 [1967]; Mann et al., Current Microbiol.13:131-135 [1986]; Holubova, Folia Microbiol. 30:97 [1985]; Chang etal., Mol. Gen. Genet. 168:11-115 [1979]; Vorobjeva et al., FEMSMicrobiol. Lett. 7:261-263 [1980]; Smith et al., Appl. Env. Microbiol.51:634 [1986]; Fisher et al., Arch. Microbiol. 139:213-217 [1981]; andMcDonald, J. Gen. Microbiol. 130:203 [1984]). Indeed, such methods astransformation, including protoplast transformation and congression,transduction, and protoplast fusion are well known and suited for use inthe present invention. Methods of transformation are used to introduce aDNA construct or vector comprising a nucleic acid encoding a variantlipolytic enzyme of the present invention into a host cell. Methodsknown in the art to transform Bacillus cells include such methods asplasmid marker rescue transformation, which involves the uptake of adonor plasmid by competent cells carrying a partially homologousresident plasmid (See, Contente et al., Plasmid 2:555-571 [1979]; Haimaet al., Mol. Gen. Genet. 223:185-191 [1990]; Weinrauch et al., J.Bacteriol. 154:1077-1087 [1983]; and Weinrauch et al., J. Bacteriol.169:1205-1211 [1987]). In this method, the incoming donor plasmidrecombines with the homologous region of the resident “helper” plasmidin a process that mimics chromosomal transformation.

In addition to commonly used methods, in some embodiments, host cellsare directly transformed with a DNA construct or vector comprising anucleic acid encoding a variant lipolytic enzyme of the invention (i.e.,an intermediate cell is not used to amplify, or otherwise process, theDNA construct or vector prior to introduction into the host cell).Introduction of the DNA construct or vector of the invention into thehost cell includes those physical and chemical methods known in the artto introduce a nucleic acid sequence (e.g., DNA sequence) into a hostcell without insertion into a plasmid or vector. Such methods include,but are not limited to calcium chloride precipitation, electroporation,naked DNA, liposomes and the like. In additional embodiments, DNAconstructs or vector are co-transformed with a plasmid, without beinginserted into the plasmid. In further embodiments, a selective marker isdeleted from the altered Bacillus strain by methods known in the art(See, Stahl et al., J. Bacteriol. 158:411-418 [1984]; and Palmeros etal., Gene 247:255-264 [2000]).

In some embodiments, the transformed cells of the present invention arecultured in conventional nutrient media. The suitable specific cultureconditions, such as temperature, pH and the like are known to thoseskilled in the art and are well described in the scientific literature.In some embodiments, the invention provides a culture (e.g., cellculture) comprising at least one variant lipolytic enzyme or at leastone nucleic acid of the invention. Also provided are compositionscomprising at least one nucleic acid, vector, or DNA construct of theinvention.

In some embodiments, host cells transformed with at least onepolynucleotide sequence encoding at least one variant lipolytic enzymeof the invention are cultured in a suitable nutrient medium underconditions permitting the expression of the present lipolytic enzyme,after which the resulting lipolytic enzyme is recovered from theculture. The medium used to culture the cells comprises any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (See e.g., the catalogues of the American Type CultureCollection). In some embodiments, the lipolytic enzyme produced by thecells is recovered from the culture medium by conventional procedures,including, but not limited to for example, separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt (e.g., ammonium sulfate), chromatographic purification (e.g., ionexchange, gel filtration, affinity, etc.). Any method suitable forrecovering or purifying a variant lipolytic enzyme finds use in thepresent invention.

In some embodiments, a variant lipolytic enzyme produced by arecombinant host cell is secreted into the culture medium. A nucleicacid sequence that encodes a purification facilitating domain may beused to facilitate purification of soluble proteins. A vector or DNAconstruct comprising a polynucleotide sequence encoding a variantlipolytic enzyme may further comprise a nucleic acid sequence encoding apurification facilitating domain to facilitate purification of thevariant lipolytic enzyme (See e.g., Kroll et al., DNA Cell Biol.12:441-53 [1993]). Such purification facilitating domains include, butare not limited to, for example, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals (See, Porath, Protein Expr. Purif. 3:263-281 [1992]), protein Adomains that allow purification on immobilized immunoglobulin, and thedomain utilized in the FLAGS extension/affinity purification system(e.g., protein A domains available from Immunex Corp., Seattle, Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (e.g., sequences available from Invitrogen, San Diego,Calif.) between the purification domain and the heterologous proteinalso find use to facilitate purification.

Assays for detecting and measuring the enzymatic activity of an enzyme,such as a variant lipolytic enzyme of the invention, are well known.Various assays for detecting and measuring activity of lipolytic enzymes(e.g., variant lipolytic enzymes of the invention), are also known tothose of ordinary skill in the art. As used herein, lipolytic activitymay be determined according to any procedure known in the art. Forexample, assays such as gel-diffusion assays of lipolysis oftriacylglycerol, titrimetry using a pH-stat method to measure release offatty acids, release of p-nitrophenol from p-Nitrophenyl esters usingspectrophotometry, and ELISA assays can be used to determine lipaseactivity and/or specificity (See, e.g. Gupta et al., Biotechnol. Appl.Biochem, 37: 63-71, 2003). Other assays can be found, for example inU.S. Pat. No. 5,990,069; and International Publication No. WO96/18729A1.

A variety of methods can be used to determine the level of production ofa mature lipolytic enzyme (e.g., mature variant lipolytic enzymes of thepresent invention) in a host cell. Such methods include, but are notlimited to, for example, methods that utilize either polyclonal ormonoclonal antibodies specific for the lipolytic enzyme. Exemplarymethods include, but are not limited to enzyme-linked immunosorbentassays (ELISA), radioimmunoassays (RIA), fluorescent immunoassays (FIA),and fluorescent activated cell sorting (FACS). These and other assaysare well known in the art (See e.g., Maddox et al., J. Exp. Med.158:1211 [1983]).

In some other embodiments, the invention provides methods for making orproducing a mature variant lipolytic enzyme of the invention. A maturevariant lipolytic enzyme does not include a signal peptide or apropeptide sequence. Some methods comprise making or producing a variantlipolytic enzyme of the invention in a recombinant bacterial host cell,such as for example, a Bacillus sp. cell (e.g., a B. subtilis cell). Insome embodiments, the invention provides a method of producing a variantlipolytic enzyme of the invention, the method comprising cultivating arecombinant host cell comprising a recombinant expression vectorcomprising a nucleic acid encoding a variant lipolytic enzyme of theinvention under conditions conducive to the production of the variantlipolytic enzyme. Some such methods further comprise recovering thevariant lipolytic enzyme from the culture.

In some embodiments the invention provides methods of producing avariant lipolytic enzyme of the invention, the methods comprising: (a)introducing a recombinant expression vector comprising a nucleic acidencoding a variant lipolytic enzyme of the invention into a populationof cells (e.g., bacterial cells, such as B. subtilis cells); and (b)culturing the cells in a culture medium under conditions conducive toproduce the variant lipolytic enzyme encoded by the expression vector.Some such methods further comprise: (c) isolating the variant lipolyticenzyme from the cells or from the culture medium.

Fabric and Home Care Products

In some embodiments, the lipolytic enzyme variants of the presentinvention can be used in compositions comprising an adjunct material anda lipolytic enzyme variant, wherein the composition is a fabric and homecare product. Examples of suitable compositions are described in Example1.

In some embodiments, the fabric and home care product compositionscomprising at least one lipolytic enzyme variant comprise one or more ofthe following ingredients (based on total composition weight): fromabout 0.0005 wt % to about 0.5 wt %, from about 0.001 wt % to about 0.1wt %, or even from about 0.002 wt % to about 0.05 wt % of said lipolyticenzyme variant; and one or more of the following: from about 0.00003 wt% to about 0.1 wt % fabric hueing agent; from about 0.001 wt % to about5 wt %, perfume capsules; from about 0.001 wt % to about 1 wt %,cold-water soluble brighteners; from about 0.00003 wt % to about 0.1 wt% bleach catalysts; from about 0.00003 wt % to about 0.1 wt % bacterialcleaning cellulases; and/or from about 0.05 wt % to about 20 wt %Guerbet nonionic surfactants.

As used herein, “wash performance” of a lipolytic enzyme (e.g., avariant lipolytic enzyme of the invention) refers to the contribution ofthe lipolytic enzyme to washing that provides additional cleaningperformance to the detergent as compared to the detergent without theaddition of the variant lipolytic enzyme to the composition. Washperformance is compared under relevant washing conditions. In some testsystems, other relevant factors, such as detergent composition, sudconcentration, water hardness, washing mechanics, time, pH, and/ortemperature, can be controlled in such a way that condition(s) typicalfor household application in a certain market segment (e.g., hand ormanual dishwashing, automatic dishwashing, dishware cleaning, tablewarecleaning, fabric cleaning, etc.) are imitated.

In some embodiments, the fabric and home care product composition is agranular or powder laundry detergent.

In some embodiments, the fabric and home care product composition is aliquid laundry detergent or a dish washing detergent.

It is intended that the fabric and home care product is provided in anysuitable form, including a fluid or solid. The fabric and home careproduct can be in the form of a unit dose pouch, especially when in theform of a liquid, and typically the fabric and home care product is atleast partially, or even completely, enclosed by a water-soluble pouch.In addition, in some embodiments of the fabric and home care productscomprising at least one lipolytic enzyme variant, the fabric and homecare product may have any combination of parameters and/orcharacteristics detailed above.

Cleaning Compositions

Cleaning compositions and cleaning formulations include any compositionthat is suited for cleaning, bleaching, disinfecting, and/or sterilizingany object, item, and/or surface. Such compositions and formulationsinclude, but are not limited to for example, liquid and/or solidcompositions, including cleaning or detergent compositions (e.g.,liquid, tablet, gel, bar, granule, and/or solid laundry cleaning ordetergent compositions and fine fabric detergent compositions; hardsurface cleaning compositions and formulations, such as for glass, wood,ceramic and metal counter tops and windows; carpet cleaners; ovencleaners; fabric fresheners; fabric softeners; and textile, laundrybooster cleaning or detergent compositions, laundry additive cleaningcompositions, and laundry pre-spotter cleaning compositions; dishwashingcompositions, including hand or manual dishwash compositions (e.g.,“hand” or “manual” dishwashing detergents) and automatic dishwashingcompositions (e.g., “automatic dishwashing detergents”).

Cleaning composition or cleaning formulations, as used herein, include,unless otherwise indicated, granular or powder-form all-purpose orheavy-duty washing agents, especially cleaning detergents; liquid,granular, gel, solid, tablet, or paste-form all-purpose washing agents,especially the so-called heavy-duty liquid (HDL) detergent or heavy-dutypowder detergent (HDD) types; liquid fine-fabric detergents; hand ormanual dishwashing agents, including those of the high-foaming type;hand or manual dishwashing, automatic dishwashing, or dishware ortableware washing agents, including the various tablet, powder, solid,granular, liquid, gel, and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car shampoos, carpet shampoos, bathroom cleaners; hairshampoos and/or hair-rinses for humans and other animals; shower gelsand foam baths and metal cleaners; as well as cleaning auxiliaries, suchas bleach additives and “stain-stick” or pre-treat types. In someembodiments, granular compositions are in “compact” form; in someembodiments, liquid compositions are in a “concentrated” form.

As used herein, the term “detergent composition” or “detergentformulation” is used in reference to a composition intended for use in awash medium for the cleaning of soiled or dirty objects, includingparticular fabric and/or non-fabric objects or items. Such compositionsof the present invention are not limited to any particular detergentcomposition or formulation. Indeed, in some embodiments, the detergentsof the invention comprise at least one variant lipolytic enzyme of theinvention and, in addition, one or more surfactants, transferase(s),hydrolytic enzymes, oxido reductases, builders (e.g., a builder salt),bleaching agents, bleach activators, bluing agents, fluorescent dyes,caking inhibitors, masking agents, enzyme activators, antioxidants,and/or solubilizers. In some instances, a builder salt is a mixture of asilicate salt and a phosphate salt, preferably with more silicate (e.g.,sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate).Some compositions of the invention, such as, but not limited to,cleaning compositions or detergent compositions, do not contain anyphosphate (e.g., phosphate salt or phosphate builder).

Unless otherwise noted, all component or composition levels providedherein are made in reference to the active level of that component orcomposition, and are exclusive of impurities, for example, residualsolvents or by-products, which may be present in commercially availablesources. Enzyme components weights are based on total active protein.All percentages and ratios are calculated by weight unless otherwiseindicated. All percentages and ratios are calculated based on the totalcomposition unless otherwise indicated. In the exemplified detergentcompositions, the enzymes levels are expressed by pure enzyme by weightof the total composition and unless otherwise specified, the detergentingredients are expressed by weight of the total compositions.

As indicated herein, in some embodiments, the cleaning compositions ofthe present invention further comprise adjunct materials including, butnot limited to, surfactants, builders, bleaches, bleach activators,bleach catalysts, other enzymes, enzyme stabilizing systems, chelants,optical brighteners, soil release polymers, dye transfer agents,dispersants, suds suppressors, dyes, perfumes, colorants, filler salts,hydrotropes, photoactivators, fluorescers, fabric conditioners,hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkageagents, anti-wrinkle agents, germicides, fungicides, color speckles,silvercare, anti-tarnish and/or anti-corrosion agents, alkalinitysources, solubilizing agents, carriers, processing aids, pigments, andpH control agents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458,5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, allof which are incorporated herein by reference). Embodiments of specificcleaning composition materials are exemplified in detail below. Inembodiments in which the cleaning adjunct materials are not compatiblewith the variant lipolytic enzymes of the present invention in thecleaning compositions, then suitable methods of keeping the cleaningadjunct materials and the lipolytic enzyme(s) separated (i.e., not incontact with each other) until combination of the two components isappropriate are used. Such separation methods include any suitablemethod known in the art (e.g., gelcaps, encapsulation, tablets, physicalseparation, etc.).

The cleaning compositions of the present invention are advantageouslyemployed for example, in laundry applications, hard surface cleaning,dishwashing applications, as well as cosmetic applications such asdentures, teeth, hair and skin. In addition, due to the uniqueadvantages of increased effectiveness in lower temperature solutions,the enzymes of the present invention are ideally suited for laundryapplications. Furthermore, the enzymes of the present invention find usein granular and liquid compositions.

The variant lipolytic enzymes of the present invention also find use incleaning additive products. In some embodiments, low temperaturesolution cleaning applications find use. In some embodiments, thepresent invention provides cleaning additive products including at leastone enzyme of the present invention is ideally suited for inclusion in awash process when additional bleaching effectiveness is desired. Suchinstances include, but are not limited to low temperature solutioncleaning applications. In some embodiments, the additive product is inits simplest form, one or more lipolytic enzymes. In some embodiments,the additive is packaged in dosage form for addition to a cleaningprocess. In some embodiments, the additive is packaged in dosage formfor addition to a cleaning process where a source of peroxygen isemployed and increased bleaching effectiveness is desired. Any suitablesingle dosage unit form finds use with the present invention, includingbut not limited to pills, tablets, gelcaps, or other single dosage unitssuch as pre-measured powders or liquids. In some embodiments, filler(s)or carrier material(s) are included to increase the volume of suchcompositions. Suitable filler or carrier materials include, but are notlimited to, various salts of sulfate, carbonate and silicate as well astalc, clay and the like. Suitable filler or carrier materials for liquidcompositions include, but are not limited to water or low molecularweight primary and secondary alcohols including polyols and diols.Examples of such alcohols include, but are not limited to, methanol,ethanol, propanol and isopropanol. In some embodiments, the compositionscontain from about 5% to about 90% of such materials. Acidic fillersfind use to reduce pH. Alternatively, in some embodiments, the cleaningadditive includes adjunct ingredients, as more fully described below.

The present cleaning compositions and cleaning additives require aneffective amount of at least one of the lipolytic enzyme variantsprovided herein, alone or in combination with other lipolytic enzymesand/or additional enzymes. The required level of enzyme is achieved bythe addition of one or more lipolytic enzyme variants of the presentinvention. Typically the present cleaning compositions comprise at leastabout 0.0001 weight percent, from about 0.0001 to about 10, from about0.001 to about 1, or even from about 0.01 to about 0.1 weight percent ofat least one of the variant lipolytic enzymes of the present invention.

The cleaning compositions herein are typically formulated such that,during use in aqueous cleaning operations, the wash water will have a pHof from about 5.0 to about 11.5, or about 6.0 to 8.0 or even from about7.5 to about 10.5. Liquid product formulations are typically formulatedto have a neat pH from about 3.0 to about 9.0 or even from about 3 toabout 8. Granular laundry products are typically formulated to have a pHfrom about 6 to about 11, or even from about 8 to about 10. Techniquesfor controlling pH at recommended usage levels include the use ofbuffers, alkalis, acids, etc., and are well known to those skilled inthe art.

Suitable “low pH cleaning compositions” typically have a neat pH of fromabout 3 to about 8, and are typically free of surfactants that hydrolyzein such a pH environment. Such surfactants include sodium alkyl sulfatesurfactants that comprise at least one ethylene oxide moiety or evenfrom about 1 to about 16 moles of ethylene oxide. Such cleaningcompositions typically comprise a sufficient amount of a pH modifier,such as sodium hydroxide, monoethanolamine or hydrochloric acid, toprovide such cleaning composition with a neat pH of from about 3 toabout 8. Such compositions typically comprise at least one acid stableenzyme. In some embodiments, the compositions are liquids, while inother embodiments, they are solids. The pH of such liquid compositionsis typically measured as a neat pH. The pH of such solid compositions ismeasured as a 10% solids solution of said composition wherein thesolvent is distilled water. In these embodiments, all pH measurementsare taken at 20° C., unless otherwise indicated.

In some embodiments, when the variant lipolytic enzyme(s) is/areemployed in a granular composition or liquid, it is desirable for thevariant lipolytic enzyme to be in the form of an encapsulated particleto protect the variant lipolytic enzyme from other components of thegranular composition during storage. In addition, encapsulation is alsoa means of controlling the availability of the variant lipolytic enzymeduring the cleaning process. In some embodiments, encapsulation enhancesthe performance of the variant lipolytic enzyme(s) and/or additionalenzymes. In this regard, the variant lipolytic enzymes of the presentinvention are encapsulated with any suitable encapsulating materialknown in the art. In some embodiments, the encapsulating materialtypically encapsulates at least part of the catalyst for the variantlipolytic enzyme(s) of the present invention. Typically, theencapsulating material is water-soluble and/or water-dispersible. Insome embodiments, the encapsulating material has a glass transitiontemperature (Tg) of 0° C. or higher. Glass transition temperature isdescribed in more detail in WO 97/11151. The encapsulating material istypically selected from consisting of carbohydrates, natural orsynthetic gums, chitin, chitosan, cellulose and cellulose derivatives,silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol,paraffin waxes, and combinations thereof. When the encapsulatingmaterial is a carbohydrate, it is typically selected frommonosaccharides, oligosaccharides, polysaccharides, and combinationsthereof. In some typical embodiments, the encapsulating material is astarch (See e.g., EP 0 922 499; U.S. Pat. Nos. 4,977,252; 5,354,559, and5,935,826). In some embodiments, the encapsulating material is amicrosphere made from plastic such as thermoplastics, acrylonitrile,methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixturesthereof; commercially available microspheres that find use include, butare not limited to those supplied by EXPANCEL® (Stockviksverken,Sweden), and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®,LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, Pa.).

As described herein, the variant lipolytic enzymes of the presentinvention find particular use in the cleaning industry, including, butnot limited to laundry and dish detergents. These applications placeenzymes under various environmental stresses. The variant lipolyticenzymes of the present invention provide advantages over many currentlyused enzymes, due to their stability under various conditions.

Indeed, there are a variety of wash conditions including varyingdetergent formulations, wash water volumes, wash water temperatures, andlengths of wash time, to which lipolytic enzymes involved in washing areexposed. In addition, detergent formulations used in differentgeographical areas have different concentrations of their relevantcomponents present in the wash water. For example, European detergentstypically have about 2000-9000 ppm of detergent components in the washwater, while Japanese detergents typically have approximately 500-1500ppm of detergent components in the wash water. In North America,particularly the United States, detergents typically have about 975 ppmof detergent components present in the wash water.

A low detergent concentration system includes detergents where less thanabout 800 ppm of the detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of the detergent components are present inthe wash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of the detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500-5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 4500 ppm to about 5000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

As a further example, different geographies use different washtemperatures. The temperature of the wash water in Japan is typicallyless than that used in Europe. For example, the temperature of the washwater in North America and Japan is typically between about 10 and about30° C. (e.g., about 20° C.), whereas the temperature of wash water inEurope is typically between about 30 and about 60° C. (e.g., about 40°C.). However, in the interest of saving energy, many consumers areswitching to using cold water washing. In addition, in some furtherregions, cold water is typically used for laundry, as well as dishwashing applications. In some embodiments, the “cold water washing” ofthe present invention utilizes “cold water detergent” suitable forwashing at temperatures from about 10° C. to about 40° C., or from about20° C. to about 30° C., or from about 15° C. to about 25° C., as well asall other combinations within the range of about 15° C. to about 35° C.,and all ranges within 10° C. to 40° C.

As a further example, different geographies typically have differentwater hardness. Water hardness is usually described in terms of thegrains per gallon mixed Ca²⁺/Mg²⁺. Hardness is a measure of the amountof calcium (Ca²⁺) and magnesium (Mg²⁺) in the water. Most water in theUnited States is hard, but the degree of hardness varies. Moderatelyhard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts permillion (parts per million converted to grains per U.S. gallon is ppm#divided by 17.1 equals grains per gallon) of hardness minerals.

Water Grains per gallon Parts per million Soft less than 1.0 less than17 Slightly hard 1.0 to 3.5 17 to 60 Moderately hard 3.5 to 7.0  60 to120 Hard  7.0 to 10.5 120 to 180 Very hard greater than 10.5 greaterthan 180

European water hardness is typically greater than about 10.5 (forexample about 10.5 to about 20.0) grains per gallon mixed Ca²⁺/Mg²⁺(e.g., about 15 grains per gallon mixed Ca²⁺/Mg²⁺). North American waterhardness is typically greater than Japanese water hardness, but lessthan European water hardness. For example, North American water hardnesscan be between about 3 to about 10 grains, about 3 to about 8 grains orabout 6 grains. Japanese water hardness is typically lower than NorthAmerican water hardness, usually less than about 4, for example about 3grains per gallon mixed Ca²⁺/Mg²⁺.

Accordingly, in some embodiments, the present invention provides variantlipolytic enzymes that show surprising wash performance in at least oneset of wash conditions (e.g., water temperature, water hardness, and/ordetergent concentration). In some embodiments, the variant lipolyticenzymes of the present invention are comparable in wash performance toother lipase lipolytic enzymes. In some embodiments, the variantlipolytic enzymes of the present invention exhibit enhanced washperformance as compared to lipase lipolytic enzymes currentlycommercially available. Thus, in some embodiments of the presentinvention, the variant lipolytic enzymes provided herein exhibitenhanced oxidative stability, enhanced thermostability, enhancedcleaning capabilities under various conditions, and/or enhanced chelatorstability. In addition, the variant lipolytic enzymes of the presentinvention find use in cleaning compositions that do not includedetergents, again either alone or in combination with builders andstabilizers.

In some embodiments of the present invention, the cleaning compositionscomprise at least one variant lipolytic enzyme of the present inventionat a level from about 0.00001% to about 10% by weight of the compositionand the balance (e.g., about 99.999% to about 90.0%) comprising cleaningadjunct materials by weight of composition. In some other embodiments ofthe present invention, the cleaning compositions of the presentinvention comprises at least one variant lipolytic enzyme at a level ofabout 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% toabout 2%, about 0.005% to about 0.5% by weight of the composition andthe balance of the cleaning composition (e.g., about 99.9999% to about90.0%, about 99.999% to about 98%, about 99.995% to about 99.5% byweight) comprising cleaning adjunct materials.

In some embodiments, the cleaning compositions of the present inventioncomprise a lipolytic enzyme variant as described above as the majorenzymatic component, such as in a mono-component composition. In someembodiments, the cleaning compositions of the present invention compriseone or more additional detergent enzymes, which provide cleaningperformance and/or fabric care and/or dishwashing benefits. Examples ofsuitable enzymes include, but are not limited to, proteases,perhydrolases, hemicellulases, cellulases, peroxidases, lipolyticenzymes, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, pectate lyases, mannanases, keratinases, reductases,oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, malanases, β-glucanases, arabinosidases,hyaluronidases, chondroitinases, laccases, and amylases, or anycombinations or mixtures thereof. In some embodiments, a combination ofenzymes is used (i. e., a “cocktail”) comprising conventional applicableenzymes like lipolytic enzyme, lipase, cutinase and/or cellulase inconjunction with amylase is used.

For example, a lipolytic enzyme variant of the invention can be combinedwith a protease. Suitable proteolytic enzymes include those of animal,vegetable or microbial origin. In some embodiments, microbialproteolytic enzymes are used. In some embodiments, the proteolyticenzyme is preferably an alkaline microbial proteolytic enzyme or atrypsin-like proteolytic enzyme. Examples of alkaline lipolytic enzymesinclude lipases, especially those derived from Bacillus (e.g., lentus,amyloliquefaciens, Carlsberg, 309, 147 and 168). Additional examplesinclude those mutant proteolytic enzymes described in U.S. Pat. Nos. RE34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which areincorporated herein by reference. Additional protease examples include,but are not limited to trypsin (e.g., of porcine or bovine origin), andthe Fusarium protease enzyme described in WO 89/06270. In someembodiments, commercially available protease enzymes that find use inthe present invention include, but are not limited to MAXATASE®,MAXACAL™, MAXAPEM™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®,PURAFECT® OXP, PURAMAX™, EXCELLASE™, and PURAFAST™ (Genencor);ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM™ POLARZYME®, OVOZYME®, KANNASE®,LIQUANASE®, NEUTRASE®, RELASE® and ESPERASE® (Novozymes); BLAP™ andBLAP™ variants (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf,Germany), and KAP (B. alkalophilus lipase; Kao Corp., Tokyo, Japan).Various proteolytic enzymes are described in WO95/23221, WO 92/21760,U.S. Pat. Publ. No. 2008/0090747, and U.S. Pat. Nos. 5,801,039,5,340,735, 5,500,364, 5,855,625, US RE 34,606, 5,955,340, 5,700,676,6,312,936, and 6,482,628, and various other patents. In some furtherembodiments, metalloprotease enzymes find use in the present invention,including but not limited to the neutral metalloprotease enzymedescribed in WO 07/044993.

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise proteases at a level fromabout 0.00001% to about 10% of protease by weight of the composition andthe balance of cleaning adjunct materials by weight of composition. Insome other embodiments of the present invention, the cleaningcompositions of the present invention also comprise proteases at a levelof about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% toabout 2%, about 0.005% to about 0.5% protease by weight of thecomposition.

In some embodiments, a lipolytic enzyme variant of the invention can becombined with an amylase. In some embodiments of the present invention,any suitable amylase finds use in the present invention. In someembodiments, any amylase (e.g., alpha and/or beta) suitable for use inalkaline solutions also find use. Suitable amylases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Amylasesthat find use in the present invention, include, but are not limited toα-amylases obtained from B. licheniformis (See e.g., GB 1,296,839).Commercially available amylases that find use in the present inventioninclude, but are not limited to DURAMYL®, TERMAMYL®, FUNGAMYL®,STAINZYME®, STAINZYME PLUS®, STAINZYME ULTRA®, and BAN™ (Novozymes), aswell as POWERASE™, RAPIDASE® and MAXAMYL® P (Genencor).

In some embodiments of the present invention, the cleaning compositionsof the present invention further comprise amylases at a level from about0.00001% to about 10% of additional amylase by weight of the compositionand the balance of cleaning adjunct materials by weight of composition.In some other embodiments of the present invention, the cleaningcompositions of the present invention also comprise amylases at a levelof about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% toabout 2%, about 0.005% to about 0.5% amylase by weight of thecomposition.

In some further embodiments, any suitable cellulase finds used in thecleaning compositions of the present invention. Suitable cellulasesinclude, but are not limited to those of bacterial or fungal origin.Chemically or genetically modified mutants are included in someembodiments. Suitable cellulases include, but are not limited toHumicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307).Especially suitable cellulases are the cellulases having color carebenefits (See e.g., EP 0 495 257). Commercially available cellulasesthat find use in the present include, but are not limited to CELLUZYME®,CAREZYME® (Novozymes), and KAC-500(B)™ (Kao Corporation). In someembodiments, cellulases are incorporated as portions or fragments ofmature wild-type or variant cellulases, wherein a portion of theN-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276). In someembodiments, the cleaning compositions of the present invention furthercomprise cellulases at a level from about 0.00001% to about 10% ofadditional cellulase by weight of the composition and the balance ofcleaning adjunct materials by weight of composition. In some otherembodiments of the present invention, the cleaning compositions of thepresent invention also comprise cellulases at a level of about 0.0001%to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about0.005% to about 0.5% cellulase by weight of the composition.

Any mannanase suitable for use in detergent compositions also finds usein the present invention. Suitable mannanases include, but are notlimited to those of bacterial or fungal origin. Chemically orgenetically modified mutants are included in some embodiments. Variousmannanases are known which find use in the present invention (See e.g.,U.S. Pat. Nos. 6,566,114, 6,602,842, and 6,440,991, all of which areincorporated herein by reference). In some embodiments, the cleaningcompositions of the present invention further comprise mannanases at alevel from about 0.00001% to about 10% of additional mannanase by weightof the composition and the balance of cleaning adjunct materials byweight of composition. In some embodiments of the present invention, thecleaning compositions of the present invention also comprise mannanasesat a level of about 0.0001% to about 10%, about 0.001% to about 5%,about 0.001% to about 2%, about 0.005% to about 0.5% mannanase by weightof the composition.

In some embodiments, peroxidases are used in combination with hydrogenperoxide or a source thereof (e.g., a percarbonate, perborate orpersulfate) in the compositions of the present invention. In somealternative embodiments, oxidases are used in combination with oxygen.Both types of enzymes are used for “solution bleaching” (i.e., toprevent transfer of a textile dye from a dyed fabric to another fabricwhen the fabrics are washed together in a wash liquor), preferablytogether with an enhancing agent (See e.g., WO 94/12621 and WO95/01426). Suitable peroxidases/oxidases include, but are not limited tothose of plant, bacterial or fungal origin. Chemically or geneticallymodified mutants are included in some embodiments. In some embodiments,the cleaning compositions of the present invention further compriseperoxidase and/or oxidase enzymes at a level from about 0.00001% toabout 10% of additional peroxidase and/or oxidase by weight of thecomposition and the balance of cleaning adjunct materials by weight ofcomposition. In some other embodiments of the present invention, thecleaning compositions of the present invention also comprise, peroxidaseand/or oxidase enzymes at a level of about 0.0001% to about 10%, about0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5%peroxidase and/or oxidase enzymes by weight of the composition.

In some embodiments, additional enzymes find use, including but notlimited to perhydrolases (See e.g., WO 05/056782). In addition, in someembodiments, mixtures of the above mentioned enzymes are encompassedherein, in particular one or more additional lipolytic enzyme, amylase,protease, mannanase, and/or at least one cellulase. Indeed, it iscontemplated that various mixtures of these enzymes will find use in thepresent invention. It is also contemplated that the varying levels ofthe variant lipolytic enzyme(s) and one or more additional enzymes mayboth independently range to about 10%, the balance of the cleaningcomposition being cleaning adjunct materials. The specific selection ofcleaning adjunct materials are readily made by considering the surface,item, or fabric to be cleaned, and the desired form of the compositionfor the cleaning conditions during use (e.g., through the wash detergentuse).

Examples of suitable cleaning adjunct materials include, but are notlimited to, surfactants, builders, bleaches, bleach activators, bleachcatalysts, other enzymes, enzyme stabilizing systems, chelants, opticalbrighteners, soil release polymers, dye transfer agents, dye transferinhibiting agents, catalytic materials, hydrogen peroxide, sources ofhydrogen peroxide, preformed peracids, polymeric dispersing agents, claysoil removal agents, structure elasticizing agents, dispersants, sudssuppressors, dyes, perfumes, colorants, filler salts, hydrotropes,photoactivators, fluorescers, fabric conditioners, fabric softeners,carriers, hydrotropes, processing aids, solvents, pigments, hydrolyzablesurfactants, preservatives, anti-oxidants, anti-shrinkage agents,anti-wrinkle agents, germicides, fungicides, color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, alkalinity sources,solubilizing agents, carriers, processing aids, pigments, and pH controlagents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464,5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of whichare incorporated herein by reference). Embodiments of specific cleaningcomposition materials are exemplified in detail below. In embodiments inwhich the cleaning adjunct materials are not compatible with the variantlipolytic enzymes of the present invention in the cleaning compositions,then suitable methods of keeping the cleaning adjunct materials and thelipolytic enzyme(s) separated (i.e., not in contact with each other)until combination of the two components is appropriate are used. Suchseparation methods include any suitable method known in the art (e.g.,gelcaps, encapsulation, tablets, physical separation, etc.).

In some embodiments, an effective amount of one or more variantlipolytic enzyme(s) provided herein is included in compositions usefulfor cleaning a variety of surfaces in need of lipid stain removal. Suchcleaning compositions include cleaning compositions for suchapplications as cleaning hard surfaces, fabrics, and dishes. Indeed, insome embodiments, the present invention provides fabric cleaningcompositions, while in other embodiments, the present invention providesnon-fabric cleaning compositions. It is intended that the presentinvention encompass detergent compositions in any form (i.e., liquid,granular, bar, semi-solid, gels, emulsions, tablets, capsules, etc.).

By way of example, several cleaning compositions wherein the variantlipolytic enzymes of the present invention find use are described ingreater detail below. In some embodiments in which the cleaningcompositions of the present invention are formulated as compositionssuitable for use in laundry machine washing method(s), the compositionsof the present invention preferably contain at least one surfactant andat least one builder compound, as well as one or more cleaning adjunctmaterials preferably selected from organic polymeric compounds,bleaching agents, additional enzymes, suds suppressors, dispersants,lime-soap dispersants, soil suspension and anti-redeposition agents andcorrosion inhibitors. In some embodiments, laundry compositions alsocontain softening agents (i.e., as additional cleaning adjunctmaterials). The compositions of the present invention also find usedetergent additive products in solid or liquid form. Such additiveproducts are intended to supplement and/or boost the performance ofconventional detergent compositions and can be added at any stage of thecleaning process. In some embodiments, the density of the laundrydetergent compositions herein ranges from about 400 to about 1200g/liter, while in other embodiments, it ranges from about 500 to about950 g/liter of composition measured at 20° C.

In embodiments formulated as compositions for use in manual dishwashingmethods, the compositions of the invention preferably contain at leastone surfactant and preferably at least one additional cleaning adjunctmaterial selected from organic polymeric compounds, suds enhancingagents, group II metal ions, solvents, hydrotropes and additionalenzymes.

In some embodiments, various cleaning compositions such as thoseprovided in U.S. Pat. No. 6,605,458, find use with the variant lipolyticenzymes of the present invention. Thus, in some embodiments, thecompositions comprising at least one variant lipolytic enzyme of thepresent invention is a compact granular fabric cleaning composition,while in other embodiments, the composition is a granular fabriccleaning composition useful in the laundering of colored fabrics, infurther embodiments, the composition is a granular fabric cleaningcomposition which provides softening through the wash capacity, inadditional embodiments, the composition is a heavy duty liquid fabriccleaning composition. In some embodiments, the compositions comprisingat least one variant lipolytic enzyme of the present invention arefabric cleaning compositions such as those described in U.S. Pat. Nos.6,610,642 and 6,376,450. In addition, the variant lipolytic enzymes ofthe present invention find use in granular laundry detergentcompositions of particular utility under European or Japanese washingconditions (See e.g., U.S. Pat. No. 6,610,642).

In some alternative embodiments, the present invention provides hardsurface cleaning compositions comprising at least one variant lipolyticenzyme provided herein. Thus, in some embodiments, the compositionscomprising at least one variant lipolytic enzyme of the presentinvention is a hard surface cleaning composition such as those describedin U.S. Pat. Nos. 6,610,642, 6,376,450, and 6,376,450.

In yet further embodiments, the present invention provides dishwashingcompositions comprising at least one variant lipolytic enzyme providedherein. Thus, in some embodiments, the compositions comprising at leastone variant lipolytic enzyme of the present invention is a hard surfacecleaning composition such as those in U.S. Pat. Nos. 6,610,642 and6,376,450. In some still further embodiments, the present inventionprovides dishwashing compositions comprising at least one variantlipolytic enzyme provided herein. In some further embodiments, thecompositions comprising at least one variant lipolytic enzyme of thepresent invention comprise oral care compositions such as those in U.S.Pat. Nos. 6,376,450, and 6,376,450. The formulations and descriptions ofthe compounds and cleaning adjunct materials contained in theaforementioned U.S. Pat. Nos. 6,376,450, 6,605,458, 6,605,458, and6,610,642, find use with the variant lipolytic enzymes provided herein.

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any process chosen by the formulator,non-limiting examples of which are described in U.S. Pat. Nos.5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. When a low pH cleaning composition is desired, the pH of suchcomposition is adjusted via the addition of a material such asmonoethanolamine or an acidic material such as HCl.

While not essential for the purposes of the present invention, thenon-limiting list of adjuncts illustrated hereinafter are suitable foruse in the instant cleaning compositions. In some embodiments, theseadjuncts are incorporated for example, to assist or enhance cleaningperformance, for treatment of the substrate to be cleaned, or to modifythe aesthetics of the cleaning composition as is the case with perfumes,colorants, dyes or the like. It is understood that such adjuncts are inaddition to the variant lipolytic enzymes of the present invention. Theprecise nature of these additional components, and levels ofincorporation thereof, will depend on the physical form of thecomposition and the nature of the cleaning operation for which it is tobe used. Suitable adjunct materials include, but are not limited to,surfactants, builders, chelating agents, dye transfer inhibiting agents,deposition aids, dispersants, additional enzymes, and enzymestabilizers, catalytic materials, bleach activators, bleach boosters,hydrogen peroxide, sources of hydrogen peroxide, preformed peracids,polymeric dispersing agents, clay soil removal/anti-redeposition agents,brighteners, suds suppressors, dyes, perfumes, structure elasticizingagents, fabric softeners, carriers, hydrotropes, processing aids and/orpigments. In addition to the disclosure below, suitable examples of suchother adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282,6,306,812, and 6,326,348, incorporated by reference. The aforementionedadjunct ingredients may constitute the balance of the cleaningcompositions of the present invention.

In some embodiments, the cleaning compositions according to the presentinvention comprise at least one surfactant and/or a surfactant systemwherein the surfactant is selected from nonionic surfactants, anionicsurfactants, cationic surfactants, ampholytic surfactants, zwitterionicsurfactants, semi-polar nonionic surfactants and mixtures thereof. Insome low pH cleaning composition embodiments (e.g., compositions havinga neat pH of from about 3 to about 5), the composition typically doesnot contain alkyl ethoxylated sulfate, as it is believed that suchsurfactant may be hydrolyzed by such compositions the acidic contents.In some embodiments, the surfactant is present at a level of from about0.1% to about 60%, while in alternative embodiments the level is fromabout 1% to about 50%, while in still further embodiments the level isfrom about 5% to about 40%, by weight of the cleaning composition.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more detergent builders or builder systems. In someembodiments incorporating at least one builder, the cleaningcompositions comprise at least about 1%, from about 3% to about 60% oreven from about 5% to about 40% builder by weight of the cleaningcomposition. Builders include, but are not limited to, the alkali metal,ammonium and alkanolammonium salts of polyphosphates, alkali metalsilicates, alkaline earth and alkali metal carbonates, aluminosilicates,polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers ofmaleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, thevarious alkali metal, ammonium and substituted ammonium salts ofpolyacetic acids such as ethylenediamine tetraacetic acid andnitrilotriacetic acid, as well as polycarboxylates such as melliticacid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid,benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, andsoluble salts thereof. Indeed, it is contemplated that any suitablebuilder will find use in various embodiments of the present invention.

In some embodiments, the builders form water-soluble hardness ioncomplexes (e.g., sequestering builders), such as citrates andpolyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphospatehexahydrate, potassium tripolyphosphate, and mixed sodium and potassiumtripolyphosphate, etc.). It is contemplated that any suitable builderwill find use in the present invention, including those known in the art(See e.g., EP 2 100 949).

In some embodiments, the cleaning compositions of the present inventioncontain at least one chelating agent. Suitable chelating agents include,but are not limited to copper, iron and/or manganese chelating agentsand mixtures thereof. In embodiments in which at least one chelatingagent is used, the cleaning compositions of the present inventioncomprise from about 0.1% to about 15% or even from about 3.0% to about10% chelating agent by weight of the subject cleaning composition.

In some still further embodiments, the cleaning compositions providedherein contain at least one deposition aid. Suitable deposition aidsinclude, but are not limited to, polyethylene glycol, polypropyleneglycol, polycarboxylate, soil release polymers such as polytelephthalicacid, clays such as kaolinite, montmorillonite, atapulgite, illite,bentonite, halloysite, and mixtures thereof.

As indicated herein, in some embodiments, anti-redeposition agents finduse in some embodiments of the present invention. In some embodiments,non-ionic surfactants find use. For example, in automatic dishwashingembodiments, non-ionic surfactants find use for surface modificationpurposes, in particular for sheeting, to avoid filming and spotting andto improve shine. These non-ionic surfactants also find use inpreventing the re-deposition of soils. In some embodiments, theanti-redeposition agent is a non-ionic surfactant as known in the art(See e.g., EP 2 100 949).

In some embodiments, the cleaning compositions of the present inventioninclude one or more dye transfer inhibiting agents. Suitable polymericdye transfer inhibiting agents include, but are not limited to,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles or mixtures thereof. In embodiments in which atleast one dye transfer inhibiting agent is used, the cleaningcompositions of the present invention comprise from about 0.0001% toabout 10%, from about 0.01% to about 5%, or even from about 0.1% toabout 3% by weight of the cleaning composition.

In some embodiments, silicates are included within the compositions ofthe present invention. In some such embodiments, sodium silicates (e.g.,sodium disilicate, sodium metasilicate, and crystalline phyllosilicates)find use. In some embodiments, silicates are present at a level of fromabout 1% to about 20%. In some embodiments, silicates are present at alevel of from about 5% to about 15% by weight of the composition.

In some still additional embodiments, the cleaning compositions of thepresent invention also contain dispersants. Suitable water-solubleorganic materials include, but are not limited to the homo- orco-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms.

In some further embodiments, the enzymes used in the cleaningcompositions are stabilized by any suitable technique. In someembodiments, the enzymes employed herein are stabilized by the presenceof water-soluble sources of calcium and/or magnesium ions in thefinished compositions that provide such ions to the enzymes. In someembodiments, the enzyme stabilizers include oligosaccharides,polysaccharides, and inorganic divalent metal salts, including alkalineearth metals, such as calcium salts. It is contemplated that varioustechniques for enzyme stabilization will find use in the presentinvention. For example, in some embodiments, the enzymes employed hereinare stabilized by the presence of water-soluble sources of zinc (II),calcium (II) and/or magnesium (II) ions in the finished compositionsthat provide such ions to the enzymes, as well as other metal ions(e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum(III), Tin (II), cobalt (II), copper (II), nickel (II), and oxovanadium(IV). Chlorides and sulfates also find use in some embodiments of thepresent invention. Examples of suitable oligosaccharides andpolysaccharides (e.g., dextrins) are known in the art (See e.g., WO07/145964). In some embodiments, reversible lipolytic enzyme inhibitorsalso find use, such as boron-containing compounds (e.g., borate,4-formyl phenyl boronic acid) and/or a tripeptide aldehyde find use tofurther improve stability, as desired.

In some embodiments, bleaches, bleach activators and/or bleach catalystsare present in the compositions of the present invention. In someembodiments, the cleaning compositions of the present invention compriseinorganic and/or organic bleaching compound(s). Inorganic bleachesinclude, but are not limited to perhydrate salts (e.g., perborate,percarbonate, perphosphate, persulfate, and persilicate salts). In someembodiments, inorganic perhydrate salts are alkali metal salts. In someembodiments, inorganic perhydrate salts are included as the crystallinesolid, without additional protection, although in some otherembodiments, the salt is coated. Any suitable salt known in the artfinds use in the present invention (See e.g., EP 2 100 949).

In some embodiments, bleach activators are used in the compositions ofthe present invention. Bleach activators are typically organic peracidprecursors that enhance the bleaching action in the course of cleaningat temperatures of 60° C. and below. Bleach activators suitable for useherein include compounds which, under perhydrolysis conditions, givealiphatic peroxoycarboxylic acids having preferably from about 1 toabout 10 carbon atoms, in particular from about 2 to about 4 carbonatoms, and/or optionally substituted perbenzoic acid. Additional bleachactivators are known in the art and find use in the present invention(See e.g., EP 2 100 949).

In addition, in some embodiments and as further described herein, thecleaning compositions of the present invention further comprise at leastone bleach catalyst. In some embodiments, the manganesetriazacyclononane and related complexes find use, as well as cobalt,copper, manganese, and iron complexes. Additional bleach catalysts finduse in the present invention (See e.g., U.S. Pat. Nos. 4,246,612,5,227,084, 4,810410, WO 99/06521, and EP 2 100 949).

In some embodiments, the cleaning compositions of the present inventioncontain one or more catalytic metal complexes. In some embodiments, ametal-containing bleach catalyst finds use. In some embodiments, themetal bleach catalyst comprises a catalyst system comprising atransition metal cation of defined bleach catalytic activity, (e.g.,copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganesecations), an auxiliary metal cation having little or no bleach catalyticactivity (e.g., zinc or aluminum cations), and a sequestrate havingdefined stability constants for the catalytic and auxiliary metalcations, particularly ethylenediaminetetraacetic acid,ethylenediaminetetra (methylenephosphonic acid) and water-soluble saltsthereof are used (See e.g., U.S. Pat. No. 4,430,243). In someembodiments, the cleaning compositions of the present invention arecatalyzed by means of a manganese compound. Such compounds and levels ofuse are well known in the art (See e.g., U.S. Pat. No. 5,576,282). Inadditional embodiments, cobalt bleach catalysts find use in the cleaningcompositions of the present invention. Various cobalt bleach catalystsare known in the art (See e.g., U.S. Pat. Nos. 5,597,936 and 5,595,967)and are readily prepared by known procedures.

In some additional embodiments, the cleaning compositions of the presentinvention include a transition metal complex of a macropolycyclic rigidligand (MRL). As a practical matter, and not by way of limitation, insome embodiments, the compositions and cleaning processes provided bythe present invention are adjusted to provide on the order of at leastone part per hundred million of the active MRL species in the aqueouswashing medium, and in some embodiments, provide from about 0.005 ppm toabout 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, andmost preferably from about 0.1 ppm to about 5 ppm, of the MRL in thewash liquor.

In some embodiments, transition-metals in the instant transition-metalbleach catalyst include, but are not limited to manganese, iron andchromium. MRLs also include, but are not limited to special ultra-rigidligands that are cross-bridged (e.g.,5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitabletransition metal MRLs are readily prepared by known procedures (Seee.g., WO 2000/32601, and U.S. Pat. No. 6,225,464).

In some embodiments, the cleaning compositions of the present inventioncomprise metal care agents. Metal care agents find use in preventingand/or reducing the tarnishing, corrosion, and/or oxidation of metals,including aluminum, stainless steel, and non-ferrous metals (e.g.,silver and copper). Suitable metal care agents include those describedin EP 2 100 949, WO 9426860 and WO 94/26859). In some embodiments, themetal care agent is a zinc salt. In some further embodiments, thecleaning compositions of the present invention comprise from about 0.1%to about 5% by weight of one or more metal care agent.

As indicated above, the cleaning compositions of the present inventionare formulated into any suitable form and prepared by any process chosenby the formulator, non-limiting examples of which are described in U.S.Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,516,448,5,489,392, and 5,486,303, all of which are incorporated herein byreference. In some embodiments in which a low pH cleaning composition isdesired, the pH of such composition is adjusted via the addition of anacidic material such as HCl.

The cleaning compositions disclosed herein of find use in cleaning asitus (e.g., a surface, item, dishware, or fabric). Typically, at leasta portion of the situs is contacted with an embodiment of the presentcleaning composition, in neat form or diluted in a wash liquor, and thenthe situs is optionally washed and/or rinsed. For purposes of thepresent invention, “washing” includes but is not limited to, scrubbing,and mechanical agitation. In some embodiments, the cleaning compositionsare typically employed at concentrations of from about 500 ppm to about15,000 ppm in solution. When the wash solvent is water, the watertemperature typically ranges from about 5° C. to about 90° C. and, whenthe situs comprises a fabric, the water to fabric mass ratio istypically from about 1:1 to about 30:1.

Compositions for Cleaning

An aspect of the present compositions and methods is a cleaningcomposition that includes a lipolytic enzyme as a component. Anlipolytic enzyme polypeptide can be used as a component in detergentcompositions for hand washing, laundry washing, dishwashing, and otherhard-surface cleaning.

In certain embodiments, a lipolytic enzyme is incorporated intodetergents at or near a concentration conventionally used for lipolyticenzyme in detergents. For example, a lipolytic enzyme polypeptide may beadded in amount corresponding to 0.00001-1 mg (calculated as pure enzymeprotein) of lipolytic enzyme per liter of wash/dishwash liquor.Exemplary formulations are provided herein, as exemplified by thefollowing:

A lipolytic enzyme polypeptide may be a component of a detergentcomposition, as the only enzyme or with other enzymes including otheramylolytic enzymes. As such, it may be included in the detergentcomposition in the form of a non-dusting granulate, a stabilized liquid,or a protected enzyme. Non-dusting granulates may be produced, e.g., asdisclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionallybe coated by methods known in the art. Examples of waxy coatingmaterials are poly(ethylene oxide) products (polyethyleneglycol, PEG)with mean molar weights of 1,000 to 20,000; ethoxylated nonylphenolshaving from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols inwhich the alcohol contains from 12 to 20 carbon atoms and in which thereare 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; andmono- and di- and triglycerides of fatty acids. Examples of film-formingcoating materials suitable for application by fluid bed techniques aregiven in, for example, GB 1483591. Liquid enzyme preparations may, forinstance, be stabilized by adding a polyol such as propylene glycol, asugar or sugar alcohol, lactic acid or boric acid according toestablished methods. Other enzyme stabilizers are known in the art.Protected enzymes may be prepared according to the method disclosed infor example EP 238 216. Polyols have long been recognized as stabilizersof proteins, as well as improving protein solubility.

The detergent composition may be in any useful form, e.g., as powders,granules, pastes, or liquid. A liquid detergent may be aqueous,typically containing up to about 70% of water and 0% to about 30% oforganic solvent. It may also be in the form of a compact gel typecontaining only about 30% water.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0% to about 50% of anionic surfactant, such aslinear alkylbenzenesulfonate (LAS); α-olefinsulfonate (AOS); alkylsulfate (fatty alcohol sulfate) (AS); alcohol ethoxysulfate (AEOS orAES); secondary alkanesulfonates (SAS); α-sulfo fatty acid methylesters; alkyl- or alkenylsuccinic acid; or soap. The composition mayalso contain 0% to about 40% of nonionic surfactant such as alcoholethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenolethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylatedfatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxyalkyl fatty acid amide (as described for example in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes, such as proteases, another amylolytic enzyme, cutinase, lipase,cellulase, pectate lyase, perhydrolase, xylanase, peroxidase, and/orlaccase in any combination.

The detergent may contain about 1% to about 65% of a detergent builderor complexing agent such as zeolite, diphosphate, triphosphate,phosphonate, citrate, nitrilotriacetic acid (NTA),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates orlayered silicates (e.g., SKS-6 from Hoechst). The detergent may also beunbuilt, i.e. essentially free of detergent builder. The enzymes can beused in any composition compatible with the stability of the enzyme.Enzymes generally can be protected against deleterious components byknown forms of encapsulation, for example, by granulation orsequestration in hydro gels. Enzymes, and specifically lipolyticenzymes, either with or without starch binding domains, can be used in avariety of compositions including laundry and dishwashing applications,surface cleaners, as well as in compositions for ethanol production fromstarch or biomass.

The detergent may comprise one or more polymers. Examples includecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system, which may comprise a H₂O₂source such as perborate or percarbonate, which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids (e.g., the amide, imide, orsulfone type peroxyacids). The bleaching system can also be an enzymaticbleaching system, for example, perhydrolase, such as that described inInternational PCT Application WO 2005/056783.

The enzymes of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., a polyol such as propylene glycolor glycerol; a sugar or sugar alcohol; lactic acid; boric acid or aboric acid derivative such as, e.g., an aromatic borate ester; and thecomposition may be formulated as described in, e.g., WO 92/19709 and WO92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, tarnish inhibiters, opticalbrighteners, or perfumes.

The pH (measured in aqueous solution at use concentration) is usuallyneutral or alkaline, e.g., pH about 7.0 to about 11.0.

Particular forms of detergent compositions for inclusion of the presentα-lipolytic enzyme are described, below. Many of these compositions canbe provided in unit dose format for ease of use. Unit dose formulationsand packaging are described in, for example, US20090209445A1,US20100081598A1, U.S. Pat. No. 7,001,878B2, EP1504994B1, WO2001085888A2,WO2003089562A1, WO2009098659A1, WO2009098660A1, WO2009112992A1,WO2009124160A1, WO2009152031A1, WO2010059483A1, WO2010088112A1,WO2010090915A1, WO2010135238A1, WO2011094687A1, WO2011094690A1,WO2011127102A1, WO2011163428A1, WO2008000567A1, WO2006045391A1,WO2006007911A1, WO2012027404A1, EP1740690B1, WO2012059336A1, U.S. Pat.No. 6,730,646B1, WO2008087426A1, WO2010116139A1, and WO2012104613A1.

Heavy Duty Liquid (HDL) Laundry Detergent Composition

Exemplary HDL laundry detergent compositions includes a detersivesurfactant (10%-40% wt/wt), including an anionic detersive surfactant(selected from a group of linear or branched or random chain,substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkylalkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkylcarboxylates, and/or mixtures thereof), and optionally non-ionicsurfactant (selected from a group of linear or branched or random chain,substituted or unsubstituted alkyl alkoxylated alcohol, for example aC₈-C₁₈ alkyl ethoxylated alcohol and/or C₆-C₁₂ alkyl phenolalkoxylates), wherein the weight ratio of anionic detersive surfactant(with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersivesurfactant is greater than 1:1. Suitable detersive surfactants alsoinclude cationic detersive surfactants (selected from a group of alkylpyridinium compounds, alkyl quarternary ammonium compounds, alkylquarternary phosphonium compounds, alkyl ternary sulphonium compounds,and/or mixtures thereof); zwitterionic and/or amphoteric detersivesurfactants (selected from a group of alkanolamine sulpho-betaines);ampholytic surfactants; semi-polar non-ionic surfactants and mixturesthereof.

The composition may optionally include, a surfactancy boosting polymerconsisting of amphiphilic alkoxylated grease cleaning polymers (selectedfrom a group of alkoxylated polymers having branched hydrophilic andhydrophobic properties, such as alkoxylated polyalkylenimines in therange of 0.05 wt %-10 wt %) and/or random graft polymers (typicallycomprising of hydrophilic backbone comprising monomers selected from thegroup consisting of: unsaturated C₁-C₆ carboxylic acids, ethers,alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleicanhydride, saturated polyalcohols such as glycerol, and mixturesthereof; and hydrophobic side chain(s) selected from the groupconsisting of: C₄-C₂₅ alkyl group, polypropylene, polybutylene, vinylester of a saturated C₁-C₆ mono-carboxylic acid, C₁-C₆ alkyl ester ofacrylic or methacrylic acid, and mixtures thereof.

The composition may include additional polymers such as soil releasepolymers (include anionically end-capped polyesters, for example SRP1,polymers comprising at least one monomer unit selected from saccharide,dicarboxylic acid, polyol and combinations thereof, in random or blockconfiguration, ethylene terephthalate-based polymers and co-polymersthereof in random or block configuration, for example Repel-o-tex SF,SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300and SRN325, Marloquest SL), anti-redeposition polymers (0.1 wt % to 10wt %, include carboxylate polymers, such as polymers comprising at leastone monomer selected from acrylic acid, maleic acid (or maleicanhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid,citraconic acid, methylenemalonic acid, and any mixture thereof,vinylpyrrolidone homopolymer, and/or polyethylene glycol, molecularweight in the range of from 500 to 100,000 Da); cellulosic polymer(including those selected from alkyl cellulose, alkyl alkoxyalkylcellulose, carboxyalkyl cellulose, alkyl carboxyalkyl cellulose examplesof which include carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixuresthereof) and polymeric carboxylate (such as maleate/acrylate randomcopolymer or polyacrylate homopolymer).

The composition may further include saturated or unsaturated fatty acid,e.g., saturated or unsaturated C₁₂-C₂₄ fatty acid (0 wt % to 10 wt %);deposition aids (examples for which include polysaccharides, e.g.,cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC),and co-polymers of DAD MAC with vinyl pyrrolidone, acrylamides,imidazoles, imidazolinium halides, and mixtures thereof, in random orblock configuration, cationic guar gum, cationic cellulose such ascationic hydoxyethyl cellulose, cationic starch, cationicpolyacylamides, and mixtures thereof.

The composition may further include dye transfer inhibiting agents,examples of which include manganese phthalocyanine, peroxidases,polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers ofN-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones andpolyvinylimidazoles and/or mixtures thereof; chelating agents, examplesof which include ethylene-diamine-tetraacetic acid (EDTA), diethylenetriamine penta methylene phosphonic acid (DTPMP), hydroxy-ethanediphosphonic acid (HEDP), ethylenediamine N,N′-disuccinic acid (EDDS),methyl glycine diacetic acid (MGDA), diethylene triamine penta aceticacid (DTPA), propylene diamine tetracetic acid (PDT A),2-hydroxypyridine-N-oxide (HPNO), or methyl glycine diacetic acid(MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamicacid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA),4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any saltsthereof, N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiaceticacid (HEIDA), dihydroxyethylglycine (DHEG),ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof.

The composition can further include enzymes (generally about 0.01 wt %active enzyme to 0.03 wt % active enzyme) selected from proteases,amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases,pectate lyases, mannanases, cutinases, laccases, phospholipases,lysophospholipases, acyltransferases, perhydrolases, arylesterases, andany mixture thereof. The composition may include an enzyme stabilizer(examples of which include polyols such as propylene glycol or glycerol,sugar or sugar alcohol, lactic acid, reversible protease inhibitor,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronicacid).

The composition optionally includes silicone or fatty-acid based sudssuppressors; heuing dyes, calcium and magnesium cations, visualsignaling ingredients, anti-foam (0.001 wt % to about 4.0 wt %), and/orstructurant/thickener (0.01 wt % to 5 wt %, selected from the groupconsisting of diglycerides and triglycerides, ethylene glycoldistearate, microcrystalline cellulose, cellulose based materials,microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixturesthereof).

The composition can be any liquid form, for example a liquid or gelform, or any combination thereof. The composition may be in any unitdose form, for example a pouch.

Heavy Duty Dry/Solid (HDD) Laundry Detergent Composition

Exemplary HDD laundry detergent compositions includes a detersivesurfactant, including anionic detersive surfactants (e.g., linear orbranched or random chain, substituted or unsubstituted alkyl sulphates,alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkylphosphonates, alkyl carboxylates and/or mixtures thereof), non-ionicdetersive surfactant (e.g., linear or branched or random chain,substituted or unsubstituted C₈-C₁₈ alkyl ethoxylates, and/or C₆-C₁₂alkyl phenol alkoxylates), cationic detersive surfactants (e.g., alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkylquaternary phosphonium compounds, alkyl ternary sulphonium compounds,and mixtures thereof), zwitterionic and/or amphoteric detersivesurfactants (e.g., alkanolamine sulpho-betaines), ampholyticsurfactants, semi-polar non-ionic surfactants, and mixtures thereof;builders including phosphate free builders (for example zeolite buildersexamples which include zeolite A, zeolite X, zeolite P and zeolite MAPin the range of 0 wt % to less than 10 wt %), phosphate builders (forexample sodium tri-polyphosphate in the range of 0 wt % to less than 10wt %), citric acid, citrate salts and nitrilotriacetic acid, silicatesalt (e.g., sodium or potassium silicate or sodium meta-silicate in therange of 0 wt % to less than 10 wt %, or layered silicate (SKS-6));carbonate salt (e.g., sodium carbonate and/or sodium bicarbonate in therange of 0 wt % to less than 80 wt %); and bleaching agents includingphotobleaches (e.g., sulfonated zinc phthalocyanines, sulfonatedaluminum phthalocyanines, xanthenes dyes, and mixtures thereof)hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoicacid or salts thereof, 3,5,5-trimethy hexanoyl oxybenzene sulfonate,tetraacetyl ethylene diamine-TAED, nonanoyloxybenzene sulfonate-NOBS,nitrile quats, and mixtures thereof), sources of hydrogen peroxide(e.g., inorganic perhydrate salts examples of which include mono ortetra hydrate sodium salt of perborate, percarbonate, persulfate,perphosphate, or persilicate), preformed hydrophilic and/or hydrophobicperacids (e.g., percarboxylic acids and salts, percarbonic acids andsalts, perimidic acids and salts, peroxymonosulfuric acids and salts,and mixtures thereof), and/or bleach catalysts (e.g., imine bleachboosters (examples of which include iminium cations and polyions),iminium zwitterions, modified amines, modified amine oxides, N-sulphonylimines, N-phosphonyl imines, N-acyl imines, thiadiazole dioxides,perfluoroimines, cyclic sugar ketones, and mixtures thereof, andmetal-containing bleach catalysts (e.g., copper, iron, titanium,ruthenium, tungsten, molybdenum, or manganese cations along with anauxiliary metal cations such as zinc or aluminum and a sequestrate suchas ethylenediaminetetraacetic acid,ethylenediaminetetra(methylenephosphonic acid), and water-soluble saltsthereof).

The composition can include enzymes, e.g., proteases, amylases, lipases,cellulases, choline oxidases, peroxidases/oxidases, pectate lyases,mannanases, cutinases, laccases, phospholipases, lysophospholipases,acyltransferase, perhydrolase, arylesterase, and any mixture thereof.

The composition may optionally include additional detergent ingredientsincluding perfume microcapsules, starch encapsulated perfume accord,hueing agents, additional polymers, including fabric integrity andcationic polymers, dye-lock ingredients, fabric-softening agents,brighteners (for example C.I. Fluorescent brighteners), flocculatingagents, chelating agents, alkoxylated polyamines, fabric depositionaids, and/or cyclodextrin.

Automatic Dishwashing (ADW) Detergent Composition

Exemplary ADW detergent composition includes non-ionic surfactants,including ethoxylated non-ionic surfactants, alcohol alkoxylatedsurfactants, epoxy-capped poly(oxyalkylated) alcohols, or amine oxidesurfactants present in amounts from 0 to 10% by weight; builders in therange of 5-60% including phosphate builders (e.g., mono-phosphates,di-phosphates, tri-polyphosphates, other oligomeric-poylphosphates,sodium tripolyphosphate-STPP) and phosphate-free builders (e.g., aminoacid-based compounds including methyl-glycine-diacetic acid (MGDA) andsalts and derivatives thereof, glutamic-N,N-diacetic acid (GLDA) andsalts and derivatives thereof, iminodisuccinic acid (IDS) and salts andderivatives thereof, carboxy methyl inulin and salts and derivativesthereof, nitrilotriacetic acid (NTA), diethylene triamine penta aceticacid (DTPA), B-alaninediacetic acid (B-ADA) and their salts,homopolymers and copolymers of poly-carboxylic acids and their partiallyor completely neutralized salts, monomeric polycarboxylic acids andhydroxycarboxylic acids and their salts in the range of 0.5% to 50% byweight; sulfonated/carboxylated polymers in the range of about 0.1% toabout 50% by weight to provide dimensional stability; drying aids in therange of about 0.1% to about 10% by weight (e.g., polyesters, especiallyanionic polyesters, optionally together with further monomers with 3 to6 functionalities—typically acid, alcohol or ester functionalities whichare conducive to polycondensation, polycarbonate-, polyurethane- and/orpolyurea-polyorganosiloxane compounds or precursor compounds, thereof,particularly of the reactive cyclic carbonate and urea type); silicatesin the range from about 1% to about 20% by weight (including sodium orpotassium silicates for example sodium disilicate, sodium meta-silicateand crystalline phyllosilicates); inorganic bleach (e.g., perhydratesalts such as perborate, percarbonate, perphosphate, persulfate andpersilicate salts) and organic bleach (e.g., organic peroxyacids,including diacyl and tetraacylperoxides, especially diperoxydodecanediocacid, diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid);bleach activators (i.e., organic peracid precursors in the range fromabout 0.1% to about 10% by weight); bleach catalysts (e.g., manganesetriazacyclononane and related complexes, Co, Cu, Mn, and Febispyridylamine and related complexes, and pentamine acetate cobalt(III)and related complexes); metal care agents in the range from about 0.1%to 5% by weight (e.g., benzatriazoles, metal salts and complexes, and/orsilicates); enzymes in the range from about 0.01 to 5.0 mg of activeenzyme per gram of automatic dishwashing detergent composition (e.g.,proteases, amylases, lipases, cellulases, choline oxidases,peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases,phospholipases, lysophospholipases, acyltransferase, perhydrolase,arylesterase, and mixtures thereof); and enzyme stabilizer components(e.g., oligosaccharides, polysaccharides, and inorganic divalent metalsalts).

Additional Detergent Compositions

Additional exemplary detergent formulations to which the presentlipolytic enzyme can be added are described, below, in the numberedparagraphs.

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 7% to about 12%; alcohol ethoxysulfate (e.g.,C₁₂₋₁₈ alcohol, 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., C₁₆₋₁₈)about 1% to about 4%; alcohol ethoxylate (e.g., C₁₄₋₁₅ alcohol, 7 EO)about 5% to about 9%; sodium carbonate (e.g., Na₂CO₃) about 14% to about20%; soluble silicate (e.g., Na₂O, 2SiO₂) about 2 to about 6%; zeolite(e.g., NaAlSiO₄) about 15% to about 22%; sodium sulfate (e.g., Na₂SO₄)0% to about 6%; sodium citrate/citric acid (e.g., C₆HsNa₃O₇/C₆HsO₇)about 0% to about 15%; sodium perborate (e.g., NaBO₃H₂O) about 11% toabout 18%; TAED about 2% to about 6%; carboxymethylcellulose (CMC) and0% to about 2%; polymers (e.g., maleic/acrylic acid, copolymer, PVP,PEG) 0-3%; enzymes (calculated as pure enzyme) 0.0001-0.1% protein; andminor ingredients (e.g., suds suppressors, perfumes, optical brightener,photobleach) 0-5%.

2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 6% to about 11%; alcohol ethoxysulfate (e.g.,C₁₂₋₁₈ alcohol, 1-2 EO) or alkyl sulfate (e.g., C₁₆₋₁₈) about 1% toabout 3%; alcohol ethoxylate (e.g., C₁₄₋₁₅ alcohol, 7 EO) about 5% toabout 9%; sodium carbonate (e.g., Na₂CO₃) about 15% to about 21%;soluble silicate (e.g., Na₂O, 2SiO₂) about 1% to about 4%; zeolite(e.g., NaAlSiO₄) about 24% to about 34%; sodium sulfate (e.g., Na₂SO₄)about 4% to about 10%; sodium citrate/citric acid (e.g.,C₆H₅Na₃O₇/C₆H₈O₇) 0% to about 15%; carboxymethylcellulose (CMC) 0% toabout 2%; polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-6%;enzymes (calculated as pure enzyme protein) 0.0001-0.1%; minoringredients (e.g., suds suppressors, perfume) 0-5%.

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 5% to about 9%; alcohol ethoxylate (e.g.,C₁₂₋₁₅ alcohol, 7 EO) about 7% to about 14%; Soap as fatty acid (e.g.,C₁₆₋₂₂ fatty acid) about 1 to about 3%; sodium carbonate (as Na₂CO₃)about 10% to about 17%; soluble silicate (e.g., Na₂O, 2SiO₂) about 3% toabout 9%; zeolite (as NaAlSiO₄) about 23% to about 33%; sodium sulfate(e.g., Na₂SO₄) 0% to about 4%; sodium perborate (e.g., NaBO₃H₂O) about8% to about 16%; TAED about 2% to about 8%; phosphonate (e.g., EDTMPA)0% to about 1%; carboxymethylcellulose (CMC) 0% to about 2%; polymers(e.g., maleic/acrylic acid copolymer, PVP, PEG) 0-3%; enzymes(calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients(e.g., suds suppressors, perfume, optical brightener) 0-5%.

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising linear alkylbenzenesulfonate(calculated as acid) about 8% to about 12%; alcohol ethoxylate (e.g.,C₁₂₋₁₅ alcohol, 7 EO) about 10% to about 25%; sodium carbonate (asNa₂CO₃) about 14% to about 22%; soluble silicate (e.g., Na₂O, 2SiO₂)about 1% to about 5%; zeolite (e.g., NaAlSiO₄) about 25% to about 35%;sodium sulfate (e.g., Na₂SO₄) 0% to about 10%; carboxymethylcellulose(CMC) 0% to about 2%; polymers (e.g., maleic/acrylic acid copolymer,PVP, PEG) 1-3%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;and minor ingredients (e.g., suds suppressors, perfume) 0-5%.

5) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 21%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO or C₁₂₋₁₅ alcohol, 5 EO)about 12% to about 18%; soap as fatty acid (e.g., oleic acid) about 3%to about 13%; alkenylsuccinic acid (C₁₂₋₁₄) 0% to about 13%;aminoethanol about 8% to about 18%; citric acid about 2% to about 8%;phosphonate 0% to about 3%; polymers (e.g., PVP, PEG) 0% to about 3%;borate (e.g., B₄O₇) 0% to about 2%; ethanol 0% to about 3%; propyleneglycol about 8% to about 14%; enzymes (calculated as pure enzymeprotein) 0.0001-0.1%; and minor ingredients (e.g., dispersants, sudssuppressors, perfume, optical brightener) 0-5%.

6) An aqueous structured liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 21%;alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol, 5 EO)3-9%; soap as fatty acid (e.g., oleic acid) about 3% to about 10%;zeolite (as NaAlSiO₄) about 14% to about 22%; potassium citrate about 9%to about 18%; borate (e.g., B₄O₇) 0% to about 2%; carboxymethylcellulose(CMC) 0% to about 2%; polymers (e.g., PEG, PVP) 0% to about 3%;anchoring polymers such as, e.g., lauryl methacrylate/acrylic acidcopolymer; molar ratio 25:1, MW 3800) 0% to about 3%; glycerol 0% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., dispersants, suds suppressors, perfume, opticalbrighteners) 0-5%.

7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising fatty alcohol sulfate about 5% toabout 10%; ethoxylated fatty acid monoethanolamide about 3% to about 9%;soap as fatty acid 0-3%; sodium carbonate (e.g., Na₂CO₃) about 5% toabout 10%; Soluble silicate (e.g., Na₂O, 2SiO₂) about 1% to about 4%;zeolite (e.g., NaAlSiO₄) about 20% to about 40%; Sodium sulfate (e.g.,Na₂SO₄) about 2% to about 8%; sodium perborate (e.g., NaBO₃H₂O) about12% to about 18%; TAED about 2% to about 7%; polymers (e.g.,maleic/acrylic acid copolymer, PEG) about 1% to about 5%; enzymes(calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients(e.g., optical brightener, suds suppressors, perfume) 0-5%.

8) A detergent composition formulated as a granulate comprising linearalkylbenzenesulfonate (calculated as acid) about 8% to about 14%;ethoxylated fatty acid monoethanolamide about 5% to about 11%; soap asfatty acid 0% to about 3%; sodium carbonate (e.g., Na₂CO₃) about 4% toabout 10%; soluble silicate (Na₂O, 2SiO₂) about 1% to about 4%; zeolite(e.g., NaAlSiO₄) about 30% to about 50%; sodium sulfate (e.g., Na₂SO₄)about 3% to about 11%; sodium citrate (e.g., C₆HsNa₃O₇) about 5% toabout 12%; polymers (e.g., PVP, maleic/acrylic acid copolymer, PEG)about 1% to about 5%; enzymes (calculated as pure enzyme protein)0.0001-0.1%; and minor ingredients (e.g., suds suppressors, perfume)0-5%.

9) A detergent composition formulated as a granulate comprising linearalkylbenzenesulfonate (calculated as acid) about 6% to about 12%;nonionic surfactant about 1% to about 4%; soap as fatty acid about 2% toabout 6%; sodium carbonate (e.g., Na₂CO₃) about 14% to about 22%;zeolite (e.g., NaAlSiO₄) about 18% to about 32%; sodium sulfate (e.g.,Na₂SO₄) about 5% to about 20%; sodium citrate (e.g., C₆H₅Na₃O₇) about 3%to about 8%; sodium perborate (e.g., NaBO₃H2O) about 4% to about 9%;bleach activator (e.g., NOBS or TAED) about 1% to about 5%;carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g.,polycarboxylate or PEG) about 1% to about 5%; enzymes (calculated aspure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., opticalbrightener, perfume) 0-5%.

10) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 15% to about 23%;alcohol ethoxysulfate (e.g., C₁₂₋₁₅ alcohol, 2-3 EO) about 8% to about15%; alcohol ethoxylate (e.g., C₁₂₋₁₅ alcohol, 7 EO, or C₁₂₋₁₅ alcohol,5 EO) about 3% to about 9%; soap as fatty acid (e.g., lauric acid) 0% toabout 3%; aminoethanol about 1% to about 5%; sodium citrate about 5% toabout 10%; hydrotrope (e.g., sodium toluensulfonate) about 2% to about6%; borate (e.g., B₄O₇) 0% to about 2%; carboxymethylcellulose 0% toabout 1%; ethanol about 1% to about 3%; propylene glycol about 2% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., polymers, dispersants, perfume, opticalbrighteners) 0-5%.

11) An aqueous liquid detergent composition comprising linearalkylbenzenesulfonate (calculated as acid) about 20% to about 32%;alcohol ethoxylate (e.g., C₁₂₋₁₅s alcohol, 7 EO, or C₁₂-1s alcohol, 5EO) 6-12%; aminoethanol about 2% to about 6%; citric acid about 8% toabout 14%; borate (e.g., B₄O₇) about 1% to about 3%; polymer (e.g.,maleic/acrylic acid copolymer, anchoring polymer such as, e.g., laurylmethacrylate/acrylic acid copolymer) 0% to about 3%; glycerol about 3%to about 8%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%;and minor ingredients (e.g., hydrotropes, dispersants, perfume, opticalbrighteners) 0-5%.

12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising anionic surfactant (linearalkylbenzenesulfonate, alkyl sulfate, α-olefinsulfonate, α-sulfo fattyacid methyl esters, alkanesulfonates, soap) about 25% to about 40%;nonionic surfactant (e.g., alcohol ethoxylate) about 1% to about 10%;sodium carbonate (e.g., Na₂CO₃) about 8% to about 25%; soluble silicates(e.g., Na₂O, 2SiO₂) about 5% to about 15%; sodium sulfate (e.g., Na₂SO₄)0% to about 5%; zeolite (NaAlSiO₄) about 15% to about 28%; sodiumperborate (e.g., NaBO₃.4H₂O) 0% to about 20%; bleach activator (TAED orNOBS) about 0% to about 5%; enzymes (calculated as pure enzyme protein)0.0001-0.1%; minor ingredients (e.g., perfume, optical brighteners)0-3%.

13) Detergent compositions as described in compositions 1)-12) supra,wherein all or part of the linear alkylbenzenesulfonate is replaced by(C₁₂-C₁₈) alkyl sulfate.

14) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising (C₁₂-C₁₈) alkyl sulfate about 9%to about 15%; alcohol ethoxylate about 3% to about 6%; polyhydroxy alkylfatty acid amide about 1% to about 5%; zeolite (e.g., NaAlSiO₄) about10% to about 20%; layered disilicate (e.g., SK56 from Hoechst) about 10%to about 20%; sodium carbonate (e.g., Na₂CO₃) about 3% to about 12%;soluble silicate (e.g., Na₂O, 2SiO₂) 0% to about 6%; sodium citrateabout 4% to about 8%; sodium percarbonate about 13% to about 22%; TAEDabout 3% to about 8%; polymers (e.g., polycarboxylates and PVP) 0% toabout 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; andminor ingredients (e.g., optical brightener, photobleach, perfume, sudssuppressors) 0-5%.

15) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/L comprising (C₁₂-C₁₈) alkyl sulfate about 4%to about 8%; alcohol ethoxylate about 11% to about 15%; soap about 1% toabout 4%; zeolite MAP or zeolite A about 35% to about 45%; sodiumcarbonate (as Na₂CO₃) about 2% to about 8%; soluble silicate (e.g.,Na₂O, 2SiO₂) 0% to about 4%; sodium percarbonate about 13% to about 22%;TAED 1-8%; carboxymethylcellulose (CMC) 0% to about 3%; polymers (e.g.,polycarboxylates and PVP) 0% to about 3%; enzymes (calculated as pureenzyme protein) 0.0001-0.1%; and minor ingredients (e.g., opticalbrightener, phosphonate, perfume) 0-3%.

16) Detergent formulations as described in 1)-15) supra, which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

17) Detergent compositions as described supra in 1), 3), 7), 9), and12), wherein perborate is replaced by percarbonate.

18) Detergent compositions as described supra in 1), 3), 7), 9), 12),14), and 15), which additionally contain a manganese catalyst. Themanganese catalyst for example is one of the compounds described in“Efficient manganese catalysts for low-temperature bleaching,” Nature369: 637-639 (1994).

19) Detergent composition formulated as a non-aqueous detergent liquidcomprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g., phosphate), anenzyme(s), and alkali. The detergent may also comprise anionicsurfactant and/or a bleach system.

As above, the present lipolytic enzyme polypeptide may be incorporatedat a concentration conventionally employed in detergents. It is atpresent contemplated that, in the detergent composition, the enzyme maybe added in an amount corresponding to 0.00001-1.0 mg (calculated aspure enzyme protein) of lipolytic enzyme polypeptide per liter of washliquor.

The detergent composition may also contain other conventional detergentingredients, e.g., deflocculant material, filler material, foamdepressors, anti-corrosion agents, soil-suspending agents, sequesteringagents, anti-soil redeposition agents, dehydrating agents, dyes,bactericides, fluorescers, thickeners, and perfumes.

The detergent composition may be formulated as a hand (manual) ormachine (automatic) laundry detergent composition, including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for manual or automatic dishwashingoperations.

Any of the cleaning compositions described, herein, may include anynumber of additional enzymes. In general the enzyme(s) should becompatible with the selected detergent, (e.g., with respect topH-optimum, compatibility with other enzymatic and non-enzymaticingredients, and the like), and the enzyme(s) should be present ineffective amounts. The following enzymes are provided as examples.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Chemically modified or protein engineered mutants areincluded, as well as naturally processed proteins. The protease may be aserine protease or a metalloprotease, an alkaline microbial protease, atrypsin-like protease, or a chymotrypsin-like protease. Examples ofalkaline proteases are subtilisins, especially those derived fromBacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309,subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples oftrypsin-like proteases are trypsin (e.g., of porcine or bovine origin),and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583).Examples of useful proteases also include but are not limited to thevariants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO98/34946. Commercially available protease enzymes include but are notlimited to: ALCALASE®, SAVINASE®, PRIMASE™, DURALASE™, ESPERASE®,KANNASE™, and BLAZE™ (Novo Nordisk A/S and Novozymes A/S); MAXATASE®,MAXACAL™, MAXAPEM™, PROPERASE®, PURAFECT®, PURAFECT OXP™, FN2™, and FN3™(Danisco US Inc.). Other exemplary proteases include NprE from Bacillusamyloliquifaciens and ASP from Cellulomonas sp. strain 69B4.

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified, proteolytically modified, or protein engineeredmutants are included. Examples of useful lipases include but are notlimited to lipases from Humicola (synonym Thermomyces), e.g., from H.lanuginosa (T. lanuginosus) (see e.g., EP 258068 and EP 305216), from H.insolens (see e.g., WO 96/13580); a Pseudomonas lipase (e.g., from P.alcaligenes or P. pseudoalcaligenes; see, e.g., EP 218 272), P. cepacia(see e.g., EP 331 376), P. stutzeri (see e.g., GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (see e.g., WO 95/06720 and WO96/27002), P. wisconsinensis (see e.g., WO 96/12012); a Bacillus lipase(e.g., from B. subtilis; see e.g., Dartois et al. Biochemica etBiophysica Acta, 1131: 253-360 (1993)), B. stearothermophilus (see e.g.,JP 64/744992), or B. pumilus (see e.g., WO 91/16422). Additional lipasevariants contemplated for use in the formulations include thosedescribed for example in: WO 92/05249, WO 94/01541, WO 95/35381, WO96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO97/04079, WO 97/07202, EP 407225, and EP 260105. Some commerciallyavailable lipase enzymes include LIPOLASE® and LIPOLASE ULTRA™ (NovoNordisk A/S and Novozymes A/S).

Polyesterases: Suitable polyesterases can be included in thecomposition, such as those described in, for example, WO 01/34899, WO01/14629, and U.S. Pat. No. 6,933,140.

Amylases: The compositions can be combined with other amylases, such asnon-production enhanced amylase. These can include commerciallyavailable amylases, such as but not limited to STAINZYME®, NATALASE®,DURAMYL®, TERMAMYL®, FUNGAMYL® and BAN™ (Novo Nordisk A/S and NovozymesA/S); RAPIDASE®, POWERASE®, and PURASTAR® (from Danisco US Inc.).

Cellulases: Cellulases can be added to the compositions. Suitablecellulases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Suitable cellulasesinclude cellulases from the genera Bacillus, Pseudomonas, Humicola,Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases producedfrom Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed for example in U.S. Pat. Nos. 4,435,307; 5,648,263;5,691,178; 5,776,757; and WO 89/09259. Exemplary cellulases contemplatedfor use are those having color care benefit for the textile. Examples ofsuch cellulases are cellulases described in for example EP 0495257, EP0531372, WO 96/11262, WO 96/29397, and WO 98/08940. Other examples arecellulase variants, such as those described in WO 94/07998; WO 98/12307;WO 95/24471; PCT/DK98/00299; EP 531315; U.S. Pat. Nos. 5,457,046;5,686,593; and 5,763,254. Commercially available cellulases includeCELLUZYME® and CAREZYME® (Novo Nordisk A/S and Novozymes A/S);CLAZINASE® and PURADAX HA® (Danisco US Inc.); and KAC-500(B)™ (KaoCorporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases contemplated for usein the compositions include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinus, e.g., from C.cinereus, and variants thereof as those described in WO 93/24618, WO95/10602, and WO 98/15257. Commercially available peroxidases includefor example GUARDZYME™ (Novo Nordisk A/S and Novozymes A/S).

The detergent composition can also comprise 2,6-β-D-fructan hydrolase,which is effective for removal/cleaning of biofilm present on householdand/or industrial textile/laundry.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additive,i.e. a separate additive or a combined additive, can be formulated e.g.,as a granulate, a liquid, a slurry, and the like. Exemplary detergentadditive formulations include but are not limited to granulates, inparticular non-dusting granulates, liquids, in particular stabilizedliquids or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (e.g., polyethyleneglycol, PEG) with mean molar weightsof 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in, forexample, GB 1483591. Liquid enzyme preparations may, for instance, bestabilized by adding a polyol such as propylene glycol, a sugar or sugaralcohol, lactic acid or boric acid according to established methods.Protected enzymes may be prepared according to the method disclosed inEP 238,216.

The detergent composition may be in any convenient form, e.g., a bar, atablet, a powder, a granule, a paste, or a liquid. A liquid detergentmay be aqueous, typically containing up to about 70% water, and 0% toabout 30% organic solvent. Compact detergent gels containing about 30%or less water are also contemplated. The detergent composition canoptionally comprise one or more surfactants, which may be non-ionic,including semi-polar and/or anionic and/or cationic and/or zwitterionic.The surfactants can be present in a wide range, from about 0.1% to about60% by weight.

When included therein the detergent will typically contain from about 1%to about 40% of an anionic surfactant, such as linearalkylbenzenesulfonate, α-olefinsulfonate, alkyl sulfate (fatty alcoholsulfate), alcohol ethoxysulfate, secondary alkanesulfonate, α-sulfofatty acid methyl ester, alkyl- or alkenylsuccinic acid, or soap.

When included therein, the detergent will usually contain from about0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl-N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0% to about 65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, triphosphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid, alkyl- or alkenylsuccinic acid, soluble silicates or layeredsilicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Exemplary polymersinclude carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA),poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylatese.g., polyacrylates, maleic/acrylic acid copolymers), and laurylmethacrylate/acrylic acid copolymers.

The enzyme(s) of the detergent composition may be stabilized usingconventional stabilizing agents, e.g., as polyol (e.g., propylene glycolor glycerol), a sugar or sugar alcohol, lactic acid, boric acid, or aboric acid derivative (e.g., an aromatic borate ester), or a phenylboronic acid derivative (e.g., 4-formylphenyl boronic acid). Thecomposition may be formulated as described in WO 92/19709 and WO92/19708.

It is contemplated that in the detergent compositions, in particular theenzyme variants, may be added in an amount corresponding to about 0.01to about 100 mg of enzyme protein per liter of wash liquor (e.g., about0.05 to about 5.0 mg of enzyme protein per liter of wash liquor or 0.1to about 1.0 mg of enzyme protein per liter of wash liquor).

Representative detergent formulations that beneficially include alipolytic enzyme polypeptide of the present invention include thedetergent formulations found in WO2013063460, pages 78-152, and inparticular the tables of pages 94 to 152 are hereby incorporated byreference. The lipolytic enzymes are normally incorporated into thedetergent composition at a level of from 0.00001% to 10% of enzymeprotein by weight of the composition. In some embodiments, the detergentcomposition comprises more than 0.0001%, 0.001%, 0.01%, or 0.1% of thelipolytic enzyme by weight of the composition. In some embodiments, thedetergent composition comprises less than 1%, 0.1%, 0.01%, or 0.001% ofthe lipolytic enzyme by weight of the composition.

Methods of Assessing Lipolytic Enzyme Activity in Detergent Compositions

Numerous lipolytic cleaning assays are known in the art, includingswatch and micro-swatch assays. The appended Examples describe only afew such assays.

In order to further illustrate the compositions and methods, andadvantages thereof, the following specific examples are given with theunderstanding that they are illustrative rather than limiting.

Processes of Making and Using Cleaning Compositions

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any suitable process chosen by theformulator, (See e.g., U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005,5,569,645, 5,565,422, 5,516,448, 5,489,392, 5,486,303, 4,515,705,4,537,706, 4,515,707, 4,550,862, 4,561,998, 4,597,898, 4,968,451,5,565,145, 5,929,022, 6,294,514 and 6,376,445).

In some embodiments, the cleaning compositions of the present inventionare provided in unit dose form, including tablets, capsules, sachets,pouches, and multi-compartment pouches. In some embodiments, the unitdose format is designed to provide controlled release of the ingredientswithin a multi-compartment pouch (or other unit dose format). Suitableunit dose and controlled release formats are known in the art (See e.g.,EP 2 100 949, WO 02/102955, U.S. Pat. Nos. 4,765,916 and 4,972,017, andWO 04/111178 for materials suitable for use in unit dose and controlledrelease formats). In some embodiments, the unit dose form is provided bytablets wrapped with a water-soluble film or water-soluble pouches.Various formats for unit doses are provided in EP 2 100 947, and areknown in the art.

Methods of Use

In some embodiments, the cleaning compositions of the present inventionfind use in cleaning surfaces (e.g., dishware), laundry, hard surfaces,contact lenses, etc. In some embodiments, at least a portion of thesurface is contacted with at least one embodiment of the cleaningcompositions of the present invention, in neat form or diluted in a washliquor, and then the surface is optionally washed and/or rinsed. Forpurposes of the present invention, “washing” includes, but is notlimited to, scrubbing, and mechanical washing. In some embodiments, thecleaning compositions of the present invention are used atconcentrations of from about 500 ppm to about 15,000 ppm in solution. Insome embodiments in which the wash solvent is water, the watertemperature typically ranges from about 5° C. to about 90° C.

The present invention provides methods for cleaning or washing an itemor surface (e.g., hard surface) in need of cleaning, including, but notlimited to methods for cleaning or washing a dishware item, a tablewareitem, a fabric item, a laundry item, personal care item, etc., or thelike, and methods for cleaning or washing a hard or soft surface (e.g.,a hard surface of an item).

In some embodiments, the present invention provides a method forcleaning an item, object, or surface in need of cleaning, the methodcomprising contacting the item or surface (or a portion of the item orsurface desired to be cleaned) with at least one variant lipaselipolytic enzyme of the present invention or a composition of thepresent invention for a sufficient time and/or under conditions suitableand/or effective to clean the item, object, or surface to a desireddegree. Some such methods further comprise rinsing the item, object, orsurface with water. For some such methods, the cleaning composition is adishwashing detergent composition and the item or object to be cleanedis a dishware item or tableware item. As used herein, a “dishware item”is an item generally used in serving or eating food. A dishware item canbe, but is not limited to for example, a dish, plate, cup, bowl, etc.,and the like. As used herein, “tableware” is a broader term thatincludes, but is not limited to for example, dishes, cutlery, knives,forks, spoons, chopsticks, glassware, pitchers, sauce boats, drinkingvessels, serving items, etc. It is intended that “tableware item”includes any of these or similar items for serving or eating food. Forsome such methods, the cleaning composition is an automatic dishwashingdetergent composition or a hand dishwashing detergent composition andthe item or object to be cleaned is a dishware or tableware item. Forsome such methods, the cleaning composition is a laundry detergentcomposition (e.g., a power laundry detergent composition or a liquidlaundry detergent composition), and the item to be cleaned is a fabricitem. In some other embodiments, the cleaning composition is a laundrypre-treatment composition.

In some embodiments, the present invention provides methods for cleaningor washing a fabric item optionally in need of cleaning or washing,respectively. In some embodiments, the methods comprise providing acomposition comprising the variant lipolytic enzyme, including but notlimited to fabric or laundry cleaning composition, and a fabric item orlaundry item in need of cleaning, and contacting the fabric item orlaundry item (or a portion of the item desired to be cleaned) with thecomposition under conditions sufficient or effective to clean or washthe fabric or laundry item to a desired degree.

In some embodiments, the present invention provides a method forcleaning or washing an item or surface (e.g., hard surface) optionallyin need of cleaning, the method comprising providing an item or surfaceto be cleaned or washed and contacting the item or surface (or a portionof the item or surface desired to be cleaned or washed) with at leastone lipase variant of the invention or a composition of the inventioncomprising at least one such lipase variant for a sufficient time and/orunder conditions sufficient or effective to clean or wash the item orsurface to a desired degree. Such compositions include, but are notlimited to for example, a cleaning composition or detergent compositionof the invention (e.g., a hand dishwashing detergent composition, handdishwashing cleaning composition, laundry detergent or fabric detergentor laundry or fabric cleaning composition, liquid laundry detergent,liquid laundry cleaning composition, powder laundry detergentcomposition, powder laundry cleaning composition, automatic dishwashingdetergent composition, laundry booster cleaning or detergentcomposition, laundry cleaning additive, and laundry pre-spottercomposition, etc.). In some embodiments, the method is repeated one ormore times, particularly if additional cleaning or washing is desired.For example, in some instance, the method optionally further comprisesallowing the item or surface to remain in contact with the at least onevariant lipolytic enzyme or composition for a period of time sufficientor effective to clean or wash the item or surface to the desired degree.In some embodiments, the methods further comprise rinsing the item orsurface with water and/or another liquid. In some embodiments, themethods further comprise contacting the item or surface with at leastone variant lipolytic enzyme of the invention or a composition of theinvention again and allowing the item or surface to remain in contactwith the at least one variant lipolytic enzyme or composition for aperiod of time sufficient to clean or wash the item or surface to thedesired degree. In some embodiments, the cleaning composition is adishwashing detergent composition and the item to be cleaned is adishware or tableware item. In some embodiments of the present methods,the cleaning composition is an automatic dishwashing detergentcomposition or a hand dishwashing detergent composition and the item tobe cleaned is a dishware or tableware item. In some embodiments of themethods, the cleaning composition is a laundry detergent composition andthe item to be cleaned is a fabric item.

The present invention also provides methods of cleaning a tableware ordishware item in an automatic dishwashing machine, the method comprisingproviding an automatic dishwashing machine, placing an amount of anautomatic dishwashing composition comprising at least one lipase variantof the present invention or a composition of the invention sufficient toclean the tableware or dishware item in the machine (e.g., by placingthe composition in an appropriate or provided detergent compartment ordispenser in the machine), putting a dishware or tableware item in themachine, and operating the machine so as to clean the tableware ordishware item (e.g., as per the manufacturer's instructions). In someembodiments, the methods include any automatic dishwashing compositiondescribed herein, which comprises, but is not limited to at least onelipase variant provided herein. The amount of automatic dishwashingcomposition to be used can be readily determined according to themanufacturer's instructions or suggestions and any form of automaticdishwashing composition comprising at least one variant lipolytic enzymeof the invention (e.g., liquid, powder, solid, gel, tablet, etc.),including any described herein, may be employed.

The present invention also provides methods for cleaning a surface, itemor object optionally in need of cleaning, the method comprisescontacting the item or surface (or a portion of the item or surfacedesired to be cleaned) with at least one variant lipase of the presentinvention or a cleaning composition of the invention in neat form ordiluted in a wash liquor for a sufficient time and/or under conditionssufficient or effective to clean or wash the item or surface to adesired degree. The surface, item, or object may then be (optionally)washed and/or rinsed if desired. For purposes of the present invention,“washing” includes, but is not limited to for example, scrubbing andmechanical agitation. In some embodiments, the cleaning compositions areemployed at concentrations of from about 500 ppm to about 15,000 ppm insolution (e.g., aqueous solution). When the wash solvent is water, thewater temperature typically ranges from about 5° C. to about 90° C. andwhen the surface, item or object comprises a fabric, the water to fabricmass ratio is typically from about 1:1 to about 30:1.

The present invention also provides methods of cleaning a laundry orfabric item in an washing machine, the method comprising providing anwashing machine, placing an amount of a laundry detergent compositioncomprising at least one variant lipase of the invention sufficient toclean the laundry or fabric item in the machine (e.g., by placing thecomposition in an appropriate or provided detergent compartment ordispenser in the machine), placing the laundry or fabric item in themachine, and operating the machine so as to clean the laundry or fabricitem (e.g., as per the manufacturer's instructions). The methods of thepresent invention include any laundry washing detergent compositiondescribed herein, comprising but not limited to at least one of anyvariant lipase provided herein. The amount of laundry detergentcomposition to be used can be readily determined according tomanufacturer's instructions or suggestions and any form of laundrydetergent composition comprising at least one variant lipolytic enzymeof the invention (e.g., solid, powder, liquid, tablet, gel, etc.),including any described herein, may be employed.

The present invention also provides methods of improved hydrolysis ofcarboxylic ester bonds, such as in long chain triglycerides. Thehydrolysis reaction can be useful in a variety of industries, includingdetergent cleaning, food, flavor industry, biocatalytic resolution ofpharmaceuticals, production of chemicals, development of biosensors,bioremediation and cosmetics and perfumes.

In some embodiments, the variants of the present invention can be usedfor transesterification in organic solvents, such as for the productionof cocoa butter equivalents (for example, through transesterification ofpalm oil) and human milk fat substitutes, and in the preparation ofglyceride products, such as in the production of butter or margarine. Insome embodiments, the variants of the present invention can be used forhydrolysis of tallow. In some embodiments, the variants of the presentinvention can be used for modifying food flavor, for example, bysynthesis of esters of short chain fatty acids and alcohols. In someembodiments, the variants of the present invention can be used forproduction of leaner meat by aiding in fat removal, or aiding in sausagefermentation, or modifying the flavor of other foods such as cheese,rice, soybean milk or apple wine (see US20110262591). In someembodiments, the variants of the present invention can be used forbaking (see WO1994004035, EP585988, EP785994)

In some embodiments, the variants of the present invention can be usedfor production of soap through hydrolysis of lipids. In someembodiments, the variants of the present invention can be used forpersonal care uses, such as cosmetics, where the variants can aid in theproduction of emollients, such as isopropyl myristate, isopropylpalmitate or 2-ethylhexylpalmitate. In some embodiments, the variants ofthe present invention can be used for aiding in digestion, treatment ofgastrointestinal disturbances, dyspepsias, and cutaneous manifestationsof digestive allergies.

In some embodiments, the variants of the present invention can be usedfor resolving racemic mixtures of pharmaceutical compounds. In someembodiments, the variants of the present invention can be used forproduction of biodiesel.

In some embodiments, the variants of the present invention can be usedfor production of biodegradable polymers, such as 1-butyl oleate ortrimethylolpropane ester.

In some embodiments, the variants of the present invention can be usedin the textile industry for removal of size lubricants, improvement ofdying properties. In some embodiments, the variants of the presentinvention can be used for degreasing, such as for degreasing of animalhides, sheepskin or wool. In some embodiments, the variants of thepresent invention can be used for increasing the pulping rate of pulp.It is also useful in control of pitch during pulp and papermanufacturing (see EP374700).

In some embodiments, the variants of the present invention can be usedfor degumming an aqueous carbohydrate solution or slurry to improve itsfilterability, particularly, a starch hydrolysate, especially a wheatstarch hydrolysate which is difficult to filter and yields cloudyfiltrates. The treatment may be performed using methods well known inthe art. See, for example, EP 219,269, EP 808,903, and U.S. Pat. No.6,103,505.

The present invention also provides methods of use in baking accordingto U.S. Pat. No. 6,558,715.

EXPERIMENTAL

The present invention is described in further detail in the followingexamples which are not in any way intended to limit the scope of theinvention as claimed.

Example 1 Methods

The following assays are standard assays used in the examples describedbelow. Occasionally specific protocols call for deviations from thesestandard assays. In those cases, deviations from these standard assayprotocols below are identified in the examples.

A. Performance Index

The performance index (PI) of an enzyme compares the performance of thevariant (measured value) and the standard enzyme (theoretical value ormeasured value) at the same protein concentration. In addition, thetheoretical values can be calculated, using the parameters of theLangmuir equation of the standard enzyme.

A performance index (PI) that is greater than 1 (PI>1) indicatesimproved performance by a variant as compared to the parent (e.g. SEQ IDNO:1) or a reference sequence (e.g. SEQ ID NO: 2, Lipex®, Novozymes),while a PI of 1 (PI=1) identifies a variant that performs the same asthe standard, and a PI that is less than 1 (PI<1) identifies a variantthat performs worse than the standard.

B. Hydrolysis of p-Nitrophenyl Esters Assay

The TLL variants are assayed for lipase activity on three differentpara-nitrophenyl (pNP) ester substrates of varying alkyl chain lengthsto determine the chain length preference of TLL variants. Table 1-1provides details of the pNP ester substrates.

TABLE 1-1 pNP Ester Substrates Substrate Abbr Chain-length Sourcep-nitrophenyl butyrate pNPB C4 Sigma (CAS 2635-84-9) p-nitrophenylcaprylate pNPO C8 Fluka (CAS 1956-10-1) (octanoate) p-nitrophenyl pNPP C16 Sigma (CAS 1492-30-4) palmitate

A reaction emulsion with pNP ester substrates is prepared using 0.8 mMpNP ester pre-suspended in ethanol (5%) in 0.05 M HEPES adjusted to pH8.2. or in 0.05 M MES, adjusted to pH 6.0. The pNP-ester/buffersuspensions are mixed and transferred to a 96-well microtiter plate(MTP) containing the enzyme sample, in a total volume of 200 μL.Dilution of the enzyme samples and their transfer volumes are adjustedto keep the reaction within a linear range. The generation of liberatedpNP is monitored over a period of 3 minutes at OD₄₀₅ nm and correctedusing blank values (no enzyme). The pNP product generated per second iscalculated using a pNP standard curve and then normalized to the addedenzyme sample in the well (mol pNP/s per added mg enzyme). When usingthe p-nitrophenyl caprylate at pH 6.0 pNP-ester/buffer suspensions aremixed and transferred to a 96-well microtiter plate (MTP) containing theenzyme sample, in a total volume of 150 μL. The plates are sealed andshaken for 10 minutes at 900 rpm at 25° C. in an iEMS shaker (Thermoscientific). After incubation 50 μl of 0.2M HEPES pH8.2 including 0.5%Triton X-100 is added. The generation of liberated pNP is read at OD₄₀₅nm and corrected using blank values (no enzyme). The performance indexfor hydrolysis is determined by comparing the hydrolysis of the variantenzyme on a particular pNP ester substrate with that of a reference TLLenzyme (SEQ ID NO: 1 or 2).

Chain length preference for lipases is determined by calculating theratio of specific activities for pairs of substrates with differentalkyl chain lengths. A ratio greater than one indicates a preference forlonger chain length when dividing the specific activity of a lipase forhydrolysis of p-nitrophenyl palmitate by its specific activity forhydrolysis of p-nitrophenyl butyrate. For the same calculation, a ratioof less than one would indicate a preference for shorter alkyl chainlength. Comparing the specific activity ratio for hydrolysis of avariant enzyme for a particular pNP ester substrate pair with that of astandard enzyme provides a measure of the level of change in alkyl chainlength preference for the TLL variants.

C. Detergent Stability Assay

Accelerated detergent stability of the TLL variants is monitored bystressing the variants in a 10% (v/v) solution of the heavy duty liquid(HDL) detergent known commercially as Tide coldwater liquid (P&G, US;heat treated) at elevated temperature.

The raw ferment of the lipases are diluted 20× with 10% (v/v) solutionof Tide coldwater liquid detergent in a 96-well PCR plate. Followingmixing, 2 μL is transferred to 96 well plate wells containing 198 μL pNPoctanoate substrate and activity is measured as described in B togenerate the unstressed value. The PCR plate is sealed and incubated ina PCR machine for 30 min at 42° C. After end incubation the plate iscooled for 3 min at 4° C. prior to measuring activity again. Activity ofthe variant enzymes is determined by transferring 4 ul of the incubatedmixtures to a 96 well plate containing 196.1 of pNP octanoate/buffersuspension, and activity is measured as described in section B togenerate the stressed value.

The performance index for detergent stability is determined by comparingthe activity ratio of stressed vs. unstressed for the variant enzymewith that of a reference TLL enzyme (SEQ ID NO:1 or 2).

D. Thermostability Assay

Accelerated thermostability of the TLL variants is monitored bystressing the variants in 50 mM HEPES, pH 8.2, with 1 ppm subtilisinBPN′-Y217L protease at elevated temperature.

95 μL of 50 mM HEPES, pH 8.2 with protease is transferred to 96-well PCRplate wells containing 5 μL of the enzyme sample. Following mixing,activity of the variant enzymes is determined by transferring 2 μl ofthe buffer/lipase mixtures to a 96 well plate containing 198 μl of pNPoctanoate/buffer suspension, and activity is measured as described insection B.

The PCR plate is sealed and incubated in a PCR machine for 30 min at 69°C. After incubation the plate is cooled at 4° C. for 3 min prior tomeasuring activity. Activity of the variant enzymes is determined bytransferring 4 ul of the incubated mixtures to a 96 well platecontaining 196.1 of pNP octanoate/buffer suspension, activity ismeasured as described in section B.

A thermostability activity ratio is calculated based on enzyme activityafter heating, divided by enzyme activity before heating, and isexpressed as percentage remaining activity. The performance index foraccelerated thermostability is determined by comparing the activityratio of the variant enzyme, with that of the similarly treatedreference TLL enzyme (SEQ ID NO:1 or 2).

E. LAS-Stability Assay

Accelerated LAS (linear alkylbenzene sulphonate, specifically sodiumdodecyl benzene sulphonate, Sigma Cat. No. 289957) stability of the TLLvariants is monitored by stressing the variants in 0.1% LAS diluted inHEPES buffer, pH 8.

80 μL of 0.1% LAS (w/v) at pH 8.2 is transferred to 96-well PCR platewells containing 20 μL of the enzyme sample. Following mixing, activityof the variant enzymes is determined by transferring 2 ul of thebuffer/lipase mixtures to a 96 well plate containing 198 ul of pNPoctanoate/buffer suspension, and activity is measured as described insection B.

The PCR plate is sealed and incubated in a PCR machine for 30 min at 25°C. After incubation the plate is cooled at 4° C. for 3 min prior tomeasuring activity. Activity of the variant enzymes is determined bytransferring 4 μl of the incubated mixtures to a 96 well platecontaining 196 μl of pNP octanoate/buffer suspension, activity ismeasured as described in section B.

A LAS-stability activity ratio is calculated based on enzyme activityafter incubation in LAS, divided by enzyme activity in the absence ofLAS, and is expressed as percentage remaining activity. The performanceindex for LAS-stability is determined by comparing the activity ratio ofthe variant enzyme, with that of the similarly treated reference TLLenzyme (SEQ ID NO:1 or 2).

F. CS-61 Microswatch Assay

Cleaning performance of the lipase variants is tested in a microswatchassay. CS-61 swatches, which are pre-stained cotton swatches stainedwith beef fat and a red dye (Center for Testmaterial, CFT, TheNetherlands) are used in a 96-well plate format. Swatches are cut into 5mm diameter pieces and placed in each well of the MTP.

The performance of the lipase variants are tested in two detergentbackgrounds, full dosage Tide coldwater liquid (heat treated for threehours at 95° C., final dosage: 0.92 g/1), and half dose Tide coldwaterliquid (heat treated for three hours at 95° C.), plus adjuvant(n-dodecyl-3-D-Maltopyranoside) (final dosage detergent: 0.46 g/L,adjuvant: 0.272 μM).

Samples of lipase variants to be tested are obtained from Milliporefiltered culture broth of cultures grown in MTP plates. The buffers usedare 20 mM HEPES (final concentration) pH 8.2 and the water hardness isadjusted to 6 gpg 2:1 Ca:Mg. A volume of 246 μl of the HDL detergentsolution (described above) is added to each swatch-containing well ofthe 96-well plate. To initiate the reaction, enzyme samples are added ata volume of 4 μL into each well. The plates are sealed and shaken for 30minutes at 900 rpm at 30° C. in an iEMS shaker (Thermo scientific).After incubation, the fabrics are rinsed 3 times with de-ionized waterusing a Hydrospeed plate washer (Tecan, Austria) and dried at 50° C.over night. Stain removal is quantified using RGB measurements of therinsed and dried fabrics, taken with a scanner (MiCrotek Scan Maker900). Images are imported into Photoshop CSII where RGB values areextracted from the swatch containing areas using IPTK 5.0 from ReindeerGraphics. Percent Soil removal (SRI) values of the washed fabric arecalculated in relation to the unwashed fabrics using the formula:% Soil Removal(SRI)=(ΔE/ΔE _(initial))*100

${{Where}\mspace{14mu}\Delta\; E} = \sqrt{\left( {R_{after} - R_{before}} \right)^{2} + \left( {G_{after} - G_{before}} \right)^{2} + \left( {B_{after} - B_{before}} \right)^{2}}$${{Where}\mspace{14mu}\Delta\; E_{initial}} = \sqrt{\left( {R_{white} - R_{before}} \right)^{2} + \left( {G_{white} - G_{before}} \right)^{2} + \left( {B_{white} - B_{before}} \right)^{2}}$

The performance index for cleaning performance is calculated bycomparing the SRI of the variant enzyme with the SRI of a reference TLLenzyme (SEQ ID NO: 1 or 2) at the same enzyme dose as the variant. ALangmuir fit is used to calculate what the SRI for the standard enzymewould be at the same enzyme dose as the variant.

G. Fabric Adhesion Assay

Adherence of the TLL variants to cotton fabric in a detergent solution(0.46 g/l heat inactivated Tide coldwater liquid detergent buffered with20 mM HEPES pH 8.2 and water hardness at a concentration of 6 gpg 2:1Ca:Mg) is tested using a microswatch assay. Bleached cotton swatches,EMPA 221, were cut into 5 mm diameter pieces and placed into each wellof a 96 well plate (3641, Corning). A volume of 95 μL of the detergentsolution is added to each swatch containing well of the plate. Toinitiate the assay, 5 μl of raw ferment is mixed into each well with 3plates run as replicate determinations. The plates are sealed and shakenfor 10 min at 900 rpm and 30° C. in an iEMS shaker (Thermo Scientific).After incubation, the microswatch plates are rinsed 3 times with tapwater using a Hydrospeed platewasher (Tecan, Austria). The level offabric bound enzyme is measured by adding 200 μL of pNP octanoate/buffersuspension to each well of the rinsed plates. Following incubation for 5min at 30° C. in an iEMS shaker (Thermo Scientific), 150 μL are thentransferred to a new 96 well plate and OD 405 nm is measured and blanksubtracted. The performance index for fabric adhesion is determined bycomparing the activity level of each TLL variant with that of thesimilarly treated reference TLL enzyme (SEQ ID NO:1 or 2).

H. Detergents

Commercially available detergent is used:

Tide coldwater liquid (P&G). Purchased commercially August 2010 and heattreated (three hours at 95° C.) to inactivate enzymes in the productformulation.

I. Protein Determination Assay

A 96-well round-bottom MTP (5042-1385, Agilent, 0.5 mL polypropylene)containing 60 μL of filtered culture broth per well was used for theHigh Performance Liquid Chromatography (HPLC) protein determinationmethod. 10 μL of each sample was injected onto an Agilent 1290 (HewlettPackard) (U)HPLC equipped with an Acquity UPLC BEH300 C41.7 μm, 2.1×50mm column (Waters) column. Protein was bound to the column using MiliQwith 0.1% trifluoroacetic acid and eluted over a gradient up to 70%acetonitrile with 0.1% trifluoroacetic acid. Absorbance was measured at220 nm and eluted peaks integrated using ChemStation software (AgilentTechnologies). The protein concentration of TLL variants was determinedbased on a standard curve of purified TLL-protein.

Example 2 Generation of TLL Variants

A. Generation of TLL Combinatorial Libraries

For production in bacillus cells, the pHYT-TLLwt plasmid DNA (describedin U.S. Patent Application No. 61/713,436) served as template to producecombinatorial libraries at pre-selected sites in the mature region (SEQID NO: 3 of U.S. Patent Application No. 61/713,436). GeneArt AG(Regensburg, Germany) was commissioned to create combinatorial librariesusing their standard protocols. The libraries consisted of transformedB. subtilis cells containing the expression plasmid encoding TLL variantsequences. The corresponding codons for each site of interest were heldas wild type or substituted with codons for at least one non-wild typeamino acid.

SEQ ID NO:3 sets forth the nucleotide sequence of the synthetic TLL gene

gctagcgcagctggcaaagaagttagccaagatctgttcaaccaattcaaccttttcgctcaatactctgcagctgcttactgcggaaagaacaacgatgcacctgctggtactaacatcacttgcacaggtaacgcatgtcctgaagtagaaaaagctgatgctacatttctttactcttttgaagatagcggcgtcggcgatgttaccggtttcttagctctggataacacaaacaaacttatcgtccttagcttcagaggctctcgctcaatcgaaaactggatcggtaaccttaattttgacttgaaagaaatcaacgatatctgctctggttgccgtggccatgacggattcacatcatcttggagaagcgtcgcagacacgcttcgccaaaaagtagaagatgccgtacgcgaacacccagattacagagtagttttcacaggtcactctcttggcggagctttagcaacagtagcaggcgctgatctccgcggtaacggatacgacattgatgtcttctcttacggcgctccgcgcgtcggtaacagagcgtttgctgaatttttaactgtacaaacaggcggaactctttatcgcatcactcacacaaacgatattgtcccgcgcttacctccgagagaatttggttactcacacagctctcctgaatactggatcaaaagcggtacattggtacctgttactcgaaacgatatcgtcaaaattgaaggaattgacgccaccggcggcaacaaccaaccgaacatccctgacatcccggcacacctttggtacttcggcttaatcggaacatgcctttaaaagctt

For production in fungal cells, DNA sequences containing variants of thecoding region for lipase from the filamentous fungus Thermomyceslanuginosus were ordered synthetically (Invitrogen, USA) carrying theattB1 and attB2 sites to allow for the Gateway® BP recombination cloninginto the pDonor221 vector (Invitrogen, USA). The pDonor-TLL plasmidswere sequence verified by the vendor. All variants were recombined viathe Gateway® LR technology with the destination vector pTTTpyr2resulting in final expression plasmids pTTTpyrG2-TLL variants. Plasmidswere generated in the Escherichia coli TOP 10 strain, purified, and usedfor fungal transformation as described further.

The expression vector contains the Trichoderma reesei cbhI promoter andterminator regions allowing strong and inducible expression of a gene ofinterest, the Aspergillus nidulans amdS and pyr2 selective markersconferring growth of transformants on minimal medium with acetamide inthe absence of uridine. The plasmids are maintained autonomously in thefungal cell due to T. reesei derived telomere regions. Usage ofreplicative plasmids results in increased frequencies of transformationand circumvents problems of locus-dependent expression observed withintegrative fungal transformation.

Using a PEG-Protoplast method a T. reesei strain deleted for majorcellulases (ΔcbhI Δcbh2 Δegl1 Δegl2 Δegl3 Δbgl1 pyr2-) was transformedwith plasmid DNA. For protoplasts preparation, spores were grown for16-24 hours at 28° C. in Trichoderma Minimal Medium MM (20 g/L glucose,15 g/L KH₂PO₄, pH 4.5, 5 g/L (NH₄)₂SO₄, 0.6 g/L MgSO₄.7H₂O, 0.6 g/LCaCl₂.2H₂O, 1 ml of 1000× T. reesei trace elements solution (5 g/LFeSO₄*7H₂O, 1.4 g/L ZnSO₄*7H₂O, 1.6 g/L MnSO₄.H₂O, 3.7 g/L CoCl₂.6H₂O))containing 5 mM uridine at shaking speed of 150 rpm. Germinating sporeswere harvested by centrifugation and treated with 50 mg/ml of GlucanexG200 (Novozymes AG, Switzerland) solution to lysate the fungal cellwalls. Further preparation of protoplasts was performed by a standardmethod, as described by Penttila et al. [Gene 61(1987) 155-164].

Transformation mixtures containing approximately 1 μg of DNA and 5×10⁶protoplasts in a total volume of 50 μl were mixed with 200 μl of 25% PEGsolution, diluted with equal volume of 1.2 M sorbitol/10 mM Tris,pH7.5/10 mM CaCl₂ solution and mixed with 1 ml of 3% agarose MinimalMedium (MM) ((20 g/L glucose, 15 g/L KH₂PO₄, 1 g/L CaCl₂*2H₂O, 1 g/LMgSO₄.7H₂O, pH 4.5, 2.5 ml/L of 400× T. reesei trace elements (175 g/Lcitric acid, 200 g/L FeSO₄*7H₂O, 16 g/L ZnSO₄.7H₂O, 3.2 g/L CuSO₄.5H₂O,1.4 g/L MnSO₄.H₂O, 0.8 g/L boric acid)) containing 1 M sorbitol and 10mM NH₄Cl. After growth of transformants, spores from each well werepooled and repatched on fresh plates with MM containing 10 mM acetamidefor additional selective pressure. Once sporulated, spores wereharvested and used for inoculation of liquid cultures either in a24-well MTP format (for screening) or shake flasks (for validationstudies) in the following production medium: 37 g/L glucose, 1 g/Lsophorose, 9 g/L casmino acids, 10 g/L (NH₄)₂SO₄, 5 g/L KH₂PO₄, 1 g/LCaCl₂.2H₂O, 1 g/L MgSO₄.7H₂O, 33 g/L 1,4-Piperazinebis(propanesulfonicacid), pH 5.5, 2.5 ml/L of 400× T. reesei trace elements). 1 ml ofproduction medium was added to produce variants in 24 well MTPs. Forshake flasks, volumes were scaled up.

Plates were grown for 6 days at 28° C. and 80% humidity with shaking at200 rpm. Culture supernatants were harvested by filtration. For largerscale fermentation and purification, spores from selected transformantswere inoculated in the 500 ml of the same production medium and grown in2 L shake flasks for 6 days at 28° C. with agitation followed byfiltration and concentration. Expression of the variants was monitoredby SDS-PAGE analysis.

The combinatorial libraries with combination of positions at whichsubstitutions were created are shown in Table 2-1 below.

TABLE 2-1 TLL libraries and combination of positions at whichsubstitutions were created Library Name Combination of positions atwhich substitutions were created in the libraries AJ1 G023 D027 F051E056 S058 L075 G091 D130 V187 I252 L264 AJ2 G023 E056 L075 D130 V187L264 AJ3 D027 D048 058 D130 D137 G163 L227 N233 L264 TG1 A018 G023 K024L075 V077 N094 D130 V154 G156 V187 TG2 Q004 N011 D027 E056 S058 I090N233 P256 TG3 A018 G023 K024 L075 N094 D111 D130 V154 G156 V187 T189L264 TG5 N011 G023 D027 E056 L075 N094 D111 D130 V154 G156 V187 T189L264 DAE1 A018 D027 P029 N033 E045 N073 L075 T189 DAE2 A018 G023 E045A049 L075 V077 G156 V187 T189

B. Generation of TLL Synthetic Gene Variants

The pHYT-TLLwt plasmid DNA (described in U.S. Patent Application No.61/713,436) served as template to produce variants, each with a fixedset of mutations at pre-selected sites in the mature region (SEQ ID NO:3 of U.S. Patent Application No. 61/713,436). GeneArt AG (Regensburg,Germany) was commissioned to create these variants using their genesynthesis technology. The variants consisted of transformed B. subtiliscells containing the expression plasmid encoding TLL variant sequences.GeneArt provided the variants in a 96 well plate, one variant per well,with the cultures frozen in glycerol. The variants generated in thismanner are listed in Table 2-2 below.

TABLE 2-2 List of TLL Synthetic Gene Variants: A018K/D027S, A018K/E045F,A018K/L075D, A018K/L075Q, A018K/T189D, D027E/G163P, D027N/E056K,D027N/N233Q, D027S/N033D, D027S/P256T, D111A/L264R, D130A/L264R,D130A/T189Q, D130A/V187N, D130A/V187T, E045F/N073R, E045F/T189D,F051T/L075R, G023K/D130A, G023K/L264R, G023Q/L075Q, K024A/L075Q,K024A/V154I, L075Q/D130A, L075Q/G156W, L075R/L264R, L227M/L264R,N011K/D027S, N033D/E045F, N073R/L075D, N094R/G156W, P029E/N033D,S058M/L264R, V187T/L264R, A018K/A049V/L075Q, A018K/A049V/V187T,A018K/D027S/E045F, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/N073R, A018K/E045F/T189D, A018K/E045F/V187T,A018K/G023K/G156W, A018K/G023K/L075Q, A018K/G023Q/L075Q,A018K/K024A/V154I, A018K/L075D/T189D, A018K/L075Q/G156W,A018K/L075Q/V187T, A018K/N033D/L075D, A018K/N033D/T189D,A018K/N073R/L075D, A018K/P029E/T189D, A018K/V154I/G156W,A049V/L075Q/T189D, A049V/V187T/T189D, D027E/S058M/G163P,D027N/N233Q/P256T, D027N/S058M/P256T, D027Q/N233Q/P256T,D027Q/S058M/P256T, D027S/L075Q/G091Q, D027S/N033D/T189D,D027S/N073R/L075D, D027S/N233Q/P256T, D027S/S058M/P256T,D111A/D130A/L264R, D130A/T189Q/L264R, D130A/V154I/G156W,D130A/V187N/L264R, D130A/V187T/L264R, E045F/L075D/T189D,E045F/N073R/L075D, E056K/D130A/L264R, E056K/D130A/T189Q,F051T/I252Q/L264R, G023K/D130A/L264R, G023K/D130A/V187T,G023K/E056K/V187T, G023K/L075R/L264R, G023K/V187N/L264R,G023K/V187T/L264R, G023Q/A049V/T189D, G023Q/D111A/L264R,G023Q/D130A/G156W, G023Q/E045F/V077I, G023Q/E056K/L075R,G023Q/K024A/G156W, G023Q/L075Q/V077I, G023Q/L075Q/V187T,G163P/L227M/L264R, I090F/N233Q/P256T, K024A/D130A/V154I,K024A/L075Q/G156W, K024A/L075Q/V077I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/D130A/V187T, L075Q/G156W/T189D,L075Q/G156W/V187N, L075Q/G156W/V187T, L075Q/N094R/V154I,L075Q/V077I/T189D, L075Q/V154I/V187T, L075Q/V187N/L264R,L075Q/V187T/L264R, L075Q/V187T/T189D, L075R/D130A/L264R,L075R/D130A/V187T, L075R/V187N/L264R, L075R/V187T/L264R,N011K/D027S/S058M, N011K/N233Q/P256T, N033D/E045F/L075D,N033D/E045F/N073R, N033D/N073R/T189D, N073R/L075D/T189D,N094R/D130A/V187T, P029E/E045F/L075D, P029E/L075D/T189D,P029E/N033D/E045F, P029E/N033D/L075D, P029E/N073R/L075D,Q004D/D027S/P256T, Q004D/N233Q/P256T, Q004D/S058M/N233Q,S058M/L227M/L264R, V077I/D130A/V154I, V077I/V187A/T189D,V187N/T189Q/L264R, A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/N073R, A018K/D027S/E045F/T189D,A018K/D027S/L075D/T189D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/E045F/A049V/G156W,A018K/E045F/A049V/V187T, A018K/E045F/L075D/T189D,A018K/E045F/L075Q/T189D, A018K/E045F/L075Q/V187T,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/T189D,A018K/G023Q/A049V/V187T, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/G156W/V187T,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V077I,A018K/G023Q/L075Q/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/G156W, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/K024A/D130A/G156W,A018K/L075Q/G156W/T189D, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D111A, A018K/L075Q/N094R/D130A,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/L075Q/V187T/T189D, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/L075D,A018K/P029E/N033D/T189D, A018K/P029E/N073R/L075D,A018K/V154I/G156W/V187T, A049V/L075Q/V077I/T189D,A049V/L075Q/V187T/T189D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/G163P/L227M/L264R,D027E/L227M/N233Q/L264R, D027E/S058M/G163P/L264R,D027N/E056K/N233Q/P256T, D027N/E056K/S058M/P256T,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027Q/S058M/I090F/P256T, D027S/D130A/I252Q/L264R,D027S/E045F/L075D/T189D, D027S/E045F/N073R/L075D,D027S/E045F/N073R/T189D, D027S/E056K/D111A/V154I,D027S/E056K/I090F/P256T, D027S/N033D/E045F/N073R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/L075D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/L075D, 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K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/D130A/V187T/T189Q/L264K,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

Example 3 Ester Hydrolysis Activity of TLL Combinatorial Variants

TLL variants created as described in Example 2 were assayed for lipaseactivity on p-nitrophenyl butyrate, p-nitrophenyl caprylate (octanoate),and p-nitrophenyl palmitate substrates as described in Example 1. Theperformance index was calculated for the variants compared to TLL (SEQID NO:1), or the reference sequence, SEQ ID NO: 2.

TABLE 3-1 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 for p-nitrophenyl butyrate hydrolysis at pH 8 are shown below.A018K/L075D, D027N/E056K, D027S/N033D, D130A/L264R, D130A/V187N,E045F/N073R, K024A/L075Q, K024A/V154I, L075Q/D130A, L075R/L264R,P029E/N033D, V187T/L264R, A018K/E045F/N073R, A018K/G023K/G156W,A018K/G023K/L075Q, A018K/L075D/T189D, A018K/V154I/G156W,A049V/L075Q/T189D, A049V/V187T/T189D, D027E/S058M/G163P,D027N/N233Q/P256T, D027S/L075Q/G091Q, D027S/N033D/T189D,D130A/V187T/L264R, E045F/L075D/T189D, E056K/D130A/T189Q,G023K/E056K/V187T, G023Q/A049V/T189D, G023Q/D111A/L264R,G023Q/L075Q/V187T, G163P/L227M/L264R, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/G156W/V187N, L075Q/V187N/L264R,L075R/D130A/L264R, L075R/D130A/V187T, L075R/V187T/L264R,N073R/L075D/T189D, P029E/N033D/E045F, V077I/D130A/V154I,V187N/T189Q/L264R, A018K/D027S/E045F/N073R, A018K/D027S/N033D/L075D,A018K/E045F/L075D/T189D, A018K/E045F/L075Q/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/V187T, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D130A, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D137Q/G163P/L227M, D027E/S058M/G163P/L264R,D027N/E056K/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/N073R/L075D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/N073R, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, G023K/D027Q/F051T/L075Q,G023K/L075Q/G156W/V187N, G023K/L075R/D130A/L264R,G023Q/A049V/L075Q/V077I, G023Q/A049V/V077I/G156W,G023Q/D027S/D111A/G156W, G023Q/K024A/L075R/G156W,G023Q/K024A/L075R/V154I, G023Q/L075Q/D130A/L264R,G023Q/V154I/G156W/V187N, K024A/L075Q/D130A/V154I,K024A/L075R/G156W/V187N, L075G/G091Q/V187N/L264R,L075Q/D130A/G156W/V187N, L075Q/V077I/G156W/V187N,N011K/D027S/E056K/P256T, N011K/E056K/N233Q/P256T,N011K/S058M/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/D027N/S058M/P256T, Q004D/D027S/I090F/P256T,Q004D/N011K/D027Q/N233Q, S058M/D137Q/G163P/N233Q,S058M/L075Q/G091Q/I252Q, A018K/A049V/L075Q/V187T/T189D,A018K/D027S/E045F/L075D/T189D, A018K/D027S/E045F/N073R/D137V,A018K/D027S/N033D/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023K/K024A/L075R/N094R, A018K/G023Q/E045F/A049V/V187T,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/L075Q/G156W, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/D130A/G156W,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/T189D, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, A018K/L075Q/N094R/G156W/V187N,A018K/P029E/N033D/N073R/L075D, A018K/V077I/G156W/V187T/T189D,D027E/S058M/D130A/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D027N/S058M/I090F/N233Q/P256T,D027S/E056K/D111A/V187N/L264R, D027S/E056K/S058M/N233Q/P256T,D027S/L075Q/D111A/D130A/V187N, D027S/L075R/V154I/T189Q/L264R,D027S/P029E/N033D/E045F/N073R, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, F051T/S058M/L075Q/G091Q/I252Q,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/E045F/A049V/L075Q/V077I, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/K024A/L075R/D130A/V154I,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/D111A/V154I/V187T,K024A/L075R/G156W/V187N/T189Q, K024A/L075R/V154I/G156W/V187Q,L075Q/D130A/V187T/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, N011K/D027N/E056K/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q, N011K/D027Q/I090F/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,P029E/N033D/E045F/L075D/T189D, Q004D/D027S/E056K/N233Q/P256T,Q004D/D027S/S058M/N233Q/P256T, Q004D/N011K/D027Q/E056K/N233Q,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/A049V/L075Q/V077I/G156W,A018K/G023Q/A049V/V077I/V187T/T189D,A018K/G023Q/E045F/A049V/G156W/V187T,A018K/G023Q/E045F/A049V/L075Q/T189D,A018K/G023Q/E045F/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156C/V187T,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N033D/E045F/N073R/L075D/T189D,A018K/N094R/D111A/D130A/V154I/V187N,A018K/P029E/N033D/E045F/L075D/T189D,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/E056K/S058M/I090F/N233Q/P256T,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/P029E/N033D/E045F/N073R/T189D,D048Q/S058M/D137Q/G163P/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/L075Q/N094R/D111A/G156W/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,K024A/L075Q/V154I/V187T/T189Q/L264R,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/N094R/D111A/G156W/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027N/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/I090F/P256T,Q004D/D027N/E056K/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/E045F/A049V/L075Q/G156W/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/N094R/G156W/V187N,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027S/E056K/L075R/D111A/G156W/V187N/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/D027S/L075Q/N094R/V154I/G156W/T189Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 3-2 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 for p-nitrophenyl butyrate hydrolysis at pH 8compared to TLL SEQ ID NO: 1 are shown below. A018K/L075D, D027S/N033D,D130A/L264R, D130A/V187N, E045F/N073R, K024A/V154I, L075R/L264R,P029E/N033D, V187T/L264R, A018K/E045F/N073R, A018K/L075D/T189D,A018K/V154I/G156W, D027E/S058M/G163P, D027S/L075Q/G091Q,D027S/N033D/T189D, D130A/V187T/L264R, G023K/E056K/V187T,G023Q/A049V/T189D, G163P/L227M/L264R, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187T/L264R, P029E/N033D/E045F, V187N/T189Q/L264R,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/L075Q/V077I/N094R, A018K/P029E/N073R/L075D,D027E/D048Q/G163P/L264R, D027E/D137Q/G163P/L227M,D027E/S058M/G163P/L264R, D027Q/E056K/N233Q/P256T,D027S/E045F/N073R/T189D, D027S/N033D/E045F/N073R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/N073R,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,G023K/D027Q/F051T/L075Q, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, G023Q/K024A/L075R/G156W,G023Q/L075Q/D130A/L264R, L075G/G091Q/V187N/L264R,N033D/E045F/N073R/T189A, P029E/N073R/L075D/T189D,Q004D/D027N/S058M/P256T, S058M/D137Q/G163P/N233Q,S058M/L075Q/G091Q/I252Q, A018K/D027S/E045F/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/V077I/D130A/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027E/S058M/D130A/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D027S/E056K/D111A/V187N/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, F051T/S058M/L075Q/G091Q/I252Q,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/G023K/D111A/G156W/L264R,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-3 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value for p-nitrophenylbutyrate hydrolysis at pH 8 compared to TLL SEQ ID NO: 1 are shownbelow. D130A/L264R, A018K/E045F/N073R, A018K/L075D/T189D,D027S/N033D/T189D, L075G/D130A/V187H, L075Q/V187N/L264R,L075R/D130A/L264R, L075R/D130A/V187T, A018K/E045F/L075D/T189D,A018K/L075Q/V077I/N094R, A018K/P029E/N073R/L075D,D027S/N033D/L075D/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,G023K/D027Q/F051T/L075Q, G023K/L075R/D130A/L264R,G023Q/K024A/L075R/G156W, L075G/G091Q/V187N/L264R,P029E/N073R/L075D/T189D, A018K/D027S/E045F/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027S/L075R/V154I/T189Q/L264R,D130A/D137Q/G163P/N233Q/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/N094R/G156W/V187N/T189Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/V154I/G156W/V187Q,N011K/G023K/D111A/G156W/L264R, A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S0581M/V187T/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-4 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 for p-nitrophenyl butyrate hydrolysis at pH 8 areshown below. A018K/L075D, D027N/E056K, D027S/N033D, D130A/L264R,D130A/V187N, E045F/N073R, K024A/L075Q, K024A/V154I, L075Q/D130A,L075R/L264R, P029E/N033D, V187T/L264R, A018K/E045F/N073R,A018K/G023K/G156W, A018K/G023K/L075Q, A018K/L075D/T189D,A018K/V154I/G156W, A049V/L075Q/T189D, A049V/V187T/T189D,D027E/S058M/G163P, D027N/N233Q/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D130A/V187T/L264R, E045F/L075D/T189D,E056K/D130A/T189Q, G023K/D130A/V187T, G023K/E056K/V187T,G023Q/A049V/T189D, G023Q/D111A/L264R, G023Q/L075Q/V187T,G163P/L227M/L264R, K024A/D130A/V154I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/G156W/V187N, L075Q/V187N/L264R,L075R/D130A/L264R, L075R/D130A/V187T, L075R/V187T/L264R,N073R/L075D/T189D, N094R/D130A/V187T, P029E/N033D/E045F,Q004D/D027S/P256T, V077I/D130A/V154I, V187N/T189Q/L264R,A018K/D027S/E045F/N073R, A018K/D027S/N033D/L075D,A018K/E045F/L075D/T189D, A018K/E045F/L075Q/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/V187T, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D130A, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D137Q/G163P/L227M, D027E/S058M/G163P/L264R,D027N/E056K/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/N073R/L075D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/N073R, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, G023K/D027Q/F051T/L075Q,G023K/K024A/V154I/V187N, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023Q/A049V/L075Q/V077I,G023Q/A049V/V077I/G156W, G023Q/D027S/D111A/G156W,G023Q/E045F/L075Q/V187T, G023Q/K024A/L075R/G156W,G023Q/K024A/L075R/V154I, G023Q/L075Q/D130A/L264R,G023Q/V154I/G156W/V187N, K024A/L075Q/D130A/V154I,K024A/L075R/G156W/V187N, L075G/G091Q/V187N/L264R,L075Q/D130A/G156W/V187N, L075Q/V077I/G156W/V187N,N011K/D027S/E056K/P256T, N011K/E056K/N233Q/P256T,N011K/S058M/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027S/I090F/P256T, Q004D/N011K/D027Q/N233Q,S058M/D137Q/G163P/N233Q, S058M/L075Q/G091Q/I252Q,A018K/A049V/L075Q/V187T/T189D, A018K/D027S/E045F/L075D/T189D,A018K/D027S/E045F/N073R/D137V, A018K/D027S/N033D/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/E045F/A049V/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/G023Q/L075Q/V187T/T189D,A018K/G023Q/V077I/D130A/G156W, A018K/G023Q/V077I/V187T/T189D,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075Q/V154I/T189D,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,A018K/L075Q/N094R/G156W/V187N, A018K/P029E/N033D/N073R/L075D,A018K/V077I/G156W/V187T/T189D, D027E/S058M/D130A/G163P/L264R,D027E/S058M/D137Q/G163P/N233Q, D027E/S058M/G163P/L227M/L264R,D027N/S058M/I090F/N233Q/P256T, D027S/E056K/D111A/V187N/L264R,D027S/E056K/S058M/N233Q/P256T, D027S/L075Q/D111A/D130A/V187N,D027S/L075R/V154I/T189Q/L264R, D027S/P029E/N033D/E045F/N073R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/G163P/L227M/L264R, D048Q/S058M/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, E045F/A049V/L075Q/V187T/T189D,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/A049V/L075Q/G156W/V187T, G023Q/E045F/A049V/L075Q/V077I,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/K024A/L075R/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/D111A/V154I/V187T, K024A/L075R/G156W/V187N/T189Q,K024A/L075R/V154I/G156W/V187Q, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075R/D111A/D130A/V187N/T189Q,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,N011K/D027N/E056K/N233Q/P256T, N011K/D027Q/E056K/S058M/N233Q,N011K/D027Q/I090F/N233Q/P256T, N011K/D027S/E056K/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, P029E/N033D/E045F/L075D/T189D,Q004D/D027S/E056K/N233Q/P256T, Q004D/D027S/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/N233Q, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/A049V/L075Q/V077I/G156W,A018K/G023Q/A049V/V077I/V187T/T189D,A018K/G023Q/E045F/A049V/G156W/V187T,A018K/G023Q/E045F/A049V/L075Q/T189D,A018K/G023Q/E045F/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156C/V187T,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N033D/E045F/N073R/L075D/T189D,A018K/N094R/D111A/D130A/V154I/V187N,A018K/P029E/N033D/E045F/L075D/T189D,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/E056K/S058M/I090F/N233Q/P256T,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/P029E/N033D/E045F/N073R/T189D,D048Q/S058M/D137Q/G163P/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/L075Q/N094R/D111A/G156W/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,K024A/L075Q/V154I/V187T/T189Q/L264R,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/N094R/D111A/G156W/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027N/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/I090F/P256T,N011K/G023Q/L075Q/D130A/V187N/T189Q,Q004D/D027N/E056K/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094RV154I/V187T/L264R,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/E045F/A049V/L075Q/G156W/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/N094R/G156W/V187N,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027S/E056K/L075R/D111A/G156W/V187N/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/D027S/L075Q/N094R/V154I/G156W/T189Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130/V154I/L264R,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 3-5 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 for p-nitrophenylbutyrate hydrolysis at pH 8 compared to SEQ ID NO: 2 are shown below.A018K/L075D, D027S/N033D, D130A/L264R, D130A/V187N, E045F/N073R,K024A/V154I, L075R/L264R, P029E/N033D, V187T/L264R, A018K/E045F/N073R,A018K/L075D/T189D, A018K/V154I/G156W, D027E/S058M/G163P,D027S/L075Q/G091Q, D027S/N033D/T189D, D130A/V187T/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023Q/A049V/T189D,G163P/L227M/L264R, K024A/D130A/V154I, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187T/L264R, N094R/D130A/V187T, P029E/N033D/E045F,Q004D/D027S/P256T, V187N/T189Q/L264R, A018K/E045F/L075D/T189D,A018K/G023K/D111A/T189Q, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D137Q/G163P/L227M, D027E/S058M/G163P/L264R,D027Q/E056K/N233Q/P256T, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/N073R, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, G023K/D027Q/F051T/L075Q,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, G023Q/K024A/L075R/G156W,G023Q/L075Q/D130A/L264R, L075G/G091Q/V187N/L264R,N033D/E045F/N073R/T189A, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,S058M/D137Q/G163P/N233Q, S058M/L075Q/G091Q/I252Q,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/K024A/L075Q/G156W, A018K/G023Q/V077I/D130A/G156W,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027E/S058M/D130A/G163P/L264R,D027E/S058M/D137Q/G163P/N233Q, D027E/S058M/G163P/L227M/L264R,D027S/E056K/D111A/V187N/L264R, D027S/L075R/V154I/T189Q/L264R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/G163P/L227M/L264R, D048Q/S058M/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, E045F/A049V/L075Q/V187T/T189D,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/V154I/G156W/V187Q,N011K/G023K/D111A/G156W/L264R, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-6 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value for p-nitrophenyl butyrate hydrolysis at pH 8 compared to SEQID NO: 2 are shown below. D130A/L264R, A018K/E045F/N073R,A018K/L075D/T189D, D027S/N033D/T189D, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,A018K/E045F/L075D/T189D, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, D027S/N033D/L075D/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, G023K/D027Q/F051T/L075Q,G023K/L075R/D130A/L264R, G023Q/K024A/L075R/G156W,L075G/G091Q/V187N/L264R, P029E/N073R/L075D/T189D,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027S/L075R/V154I/T189Q/L264R, D130A/D137Q/G163P/N233Q/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/N094R/G156W/V187N/T189Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/G023K/D111A/G156W/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-7 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 for p-nitrophenyl caprylate (octanoate) hydrolysis at pH 8 areshown below. A018K/L075Q, D027S/N033D, D130A/L264R, K024A/L075Q,L227M/L264R, P029E/N033D, A018K/G023K/L075Q, A018K/G023Q/L075Q,A018K/L075D/T189D, A018K/L075Q/G156W, A018K/L075Q/V187T,A018K/N073R/L075D, A018K/P029E/T189D, A049V/V187T/T189D,D027S/N033D/T189D, E045F/N073R/L075D, G023K/V187N/L264R,G023Q/L075Q/V187T, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/D130A/V187T, L075Q/G156W/T189D, L075Q/G156W/V187N,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,N073R/L075D/T189D, P029E/N033D/E045F, V077I/V187A/T189D,A018K/A049V/L075Q/T189D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/E045F/L075D/T189D,A018K/E045F/L075Q/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/E045F/V187T, A018K/G023Q/G156W/V187T,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D111A, A018K/L075Q/V077I/N094R,A018K/P029E/N033D/T189D, D027E/D137Q/G163P/L227M,D027E/L227M/N233Q/L264R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D027Q/F051T/L075Q, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023Q/E045F/A049V/T189D,G023Q/E045F/L075Q/G156W, G023Q/K024A/L075R/V154I,K024A/L075Q/D130A/G156W, K024A/L075Q/V187T/T189Q,K024A/L075R/G156W/V187N, L075Q/D130A/G156W/V187N,L075Q/V077I/D130A/V187Q, L075Q/V077I/V187T/T189D,L075R/D130A/V154I/L264R, N011K/D027S/E056K/P256T,N011K/S058M/N233Q/P256T, P029E/E045F/L075D/T189D,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/N011K/D027N/P256T, A018K/D027S/N033D/L075D/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D027S/P029E/N073R/L075D, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/A049V/L075Q/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/G156W, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,A018K/L075Q/N094R/G156W/V187N, A018K/L075Q/V077I/N094R/G156W,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/S058M/G163P/N233Q,D027E/D130A/G163P/N233Q/L264R, D027S/E056K/S058M/N233Q/P256T,D027S/L075R/V154I/T189Q/L264R, D027S/P029E/N033D/E045F/N073R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075Q/G156W/T189Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/V077I/D130A/G156W, G023K/N094R/G156W/V187N/T189Q,G023Q/A049V/L075Q/G156W/V187T, G023Q/A049V/L075Q/V187T/T189D,G023Q/D027S/V154I/V187N/L264R, G023Q/E045F/L075Q/V077I/V187T,G023Q/K024A/L075Q/G156W/V187Q, G023Q/K024A/L075Q/V077I/V187Q,G023Q/K024A/V077I/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,G023Q/L075Q/V077I/G156W/V187N, K024A/L075R/V154I/G156W/V187Q,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,N011K/D027N/E056K/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,N011K/G023K/E056K/L075Q/T189Q, A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/L075Q/V077I/V187T/T189D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/P029E/N033D/E045F/N073R/T189D,D048Q/S058M/D130A/G163P/N233Q/L264R,E045F/A049V/L075Q/V077I/V187T/T189D,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/E045F/A049V/L075Q/V077I/V187T,G023Q/E045F/L075Q/V077I/G156W/V187T,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,K024A/L075Q/V154I/V187N/T189Q/L264R,K024A/L075Q/V154I/V187T/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027N/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,N011K/E056K/L075Q/D130A/V187N/T189Q,Q004D/D027N/E056K/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/G156W,A018K/G023K/K024A/L075R/N094R/D130A/V1541,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/E045F/A049V/L075Q/G156W/V187T,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D111A/G156W/V187N/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/V154I/V187T/T189Q/L264R,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D111A/V154I/G156W/V187N,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/K024A/L075R/D111A/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 3-8 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 for p-nitrophenyl caprylate (octanoate) hydrolysisat pH 8 compared to TLL SEQ ID NO: 1 are shown below. D027S/N033D,D130A/L264R, L227M/L264R, P029E/N033D, A018K/L075D/T189D,A018K/P029E/T189D, D027S/N033D/T189D, G023K/V187N/L264R,L075G/D130A/V187H, L075Q/V187N/L264R, L075R/D130A/L264R,L075R/D130A/V187T, P029E/N033D/E045F, A018K/D027S/P029E/T189D,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/L075Q/V077I/N094R, D027E/D137Q/G163P/L227M,D027E/L227M/N233Q/L264R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D027Q/F051T/L075Q, G023K/L075R/D130A/L264R,P029E/N073R/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027E/D130A/G163P/N233Q/L264R,D027S/L075R/V154I/T189Q/L264R, D130A/D137Q/G163P/N233Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/N094R/G156W/V187N/T189Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/G023K/D111A/G156W/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/V077I/N094R/D130A/V187N,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-9 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value for p-nitrophenylcaprylate (octanoate) hydrolysis at pH 8 compared to TLL SEQ ID NO: 1are shown below. L227M/L264R, A018K/L075D/T189D, A018K/P029E/T189D,D027S/N033D/T189D, G023K/V187N/L264R, L075R/D130A/V187T,A018K/D027S/P029E/T189D, A018K/L075Q/V077I/N094R,D027E/L227M/N233Q/L264R, D027S/N033D/L075D/T189D,D027S/P029E/E045F/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, G023K/L075R/D130A/L264R,P029E/N073R/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027E/D130A/G163P/N233Q/L264R, D027S/L075R/V154I/T189Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/N094R/G156W/V187N/T189Q,K024A/L075R/V154I/G156W/V187Q, A018K/D027S/P029E/N033D/E045F/L075D,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-10 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 for p-nitrophenyl caprylate (octanoate) hydrolysisat pH 8 are shown below. A018K/L075Q, D130A/L264R, K024A/L075Q,L227M/L264R, A018K/G023K/L075Q, A018K/G023Q/L075Q, A018K/L075D/T189D,A018K/L075Q/G156W, A018K/L075Q/V187T, A018K/N073R/L075D,A018K/P029E/T189D, A049V/V187T/T189D, D027S/N033D/T189D,E045F/N073R/L075D, G023K/V187N/L264R, G023Q/L075Q/V187T,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075Q/G156W/T189D, L075Q/G156W/V187N, L075R/D130A/L264R,L075R/D130A/V187T, N073R/L075D/T189D, V077I/V187A/T189D,A018K/A049V/L075Q/T189D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/E045F/L075Q/V187T,A018K/G023K/D111A/T189Q, A018K/G023Q/L075Q/V077I,A018K/G023Q/L075Q/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V187T/T189D, A018K/L075Q/N094R/D111A,A018K/L075Q/V077I/N094R, A018K/P029E/N033D/T189D,D027E/L227M/N233Q/L264R, D027S/N033D/L075D/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D027Q/F051T/L075Q, G023K/L075R/D130A/L264R,G023Q/E045F/L075Q/G156W, G023Q/K024A/L075R/V154I,K024A/L075Q/D130A/G156W, K024A/L075Q/V187T/T189Q,K024A/L075R/G156W/V187N, L075Q/D130A/G156W/V187N,L075Q/V077I/D130A/V187Q, L075Q/V077I/V187T/T189D,L075R/D130A/V154I/L264R, P029E/E045F/L075D/T189D,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/N011K/D027N/P256T, A018K/D027S/N033D/L075D/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D027S/P029E/N073R/L075D, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/K024A/L075Q/V154I/G156W,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, A018K/L075Q/N094R/G156W/V187N,A018K/L075Q/V077I/N094R/G156W, A018K/P029E/N033D/N073R/L075D,D027E/D048Q/S058M/G163P/N233Q, D027E/D130A/G163P/N233Q/L264R,D027S/L075R/V154I/T189Q/L264R, D027S/P029E/N033D/E045F/N073R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075Q/G156W/T189Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/V077I/D130A/G156W, G023K/N094R/G156W/V187N/T189Q,G023Q/A049V/L075Q/G156W/V187T, G023Q/A049V/L075Q/V187T/T189D,G023Q/E045F/L075Q/V077I/V187T, G023Q/K024A/L075Q/G156W/V187Q,G023Q/K024A/L075Q/V077I/V187Q, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/V077I/G156W/V187N, K024A/L075R/V154I/G156W/V187Q,L075R/D130A/V187T/T189Q/L264R, N011K/D027N/E056K/N233Q/P256T,N011K/G023K/E056K/L075Q/T189Q, A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/L075Q/V077I/V187T/T189D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D048Q/S058M/D130A/G163P/N233Q/L264R,E045F/A049V/L075Q/V077I/V187T/T189D,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/V154I/V187N/T189Q/L264R,K024A/L075Q/V154I/V187T/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/G156W,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D111A/G156W/V187N/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/V154I/V187T/T189Q/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/V154I/G156W/V187N,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/K024A/L075R/D111A/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 3-11 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 for p-nitrophenylcaprylate (octanoate) hydrolysis at pH 8 compared to SEQ ID NO: 2 areshown below. D130A/L264R, L227M/L264R, A018K/L075D/T189D,A018K/P029E/T189D, D027S/N033D/T189D, G023K/V187N/L264R,L075G/D130A/V187H, L075R/D130A/L264R, L075R/D130A/V187T,A018K/D027S/P029E/T189D, A018K/G023K/D111A/T189Q,A018K/L075Q/V077I/N094R, D027E/L227M/N233Q/L264R,D027S/N033D/L075D/T189D, D027S/P029E/E045F/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, G023K/D027Q/F051T/L075Q,G023K/L075R/D130A/L264R, P029E/N073R/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027E/D130A/G163P/N233Q/L264R, D027S/L075R/V154I/T189Q/L264R,D130A/D137Q/G163P/N233Q/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/N094R/G156W/V187N/T189Q, G023Q/K024A/L075Q/G156W/V187Q,K024A/L075R/V154I/G156W/V187Q, A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-12 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value for p-nitrophenyl caprylate (octanoate) hydrolysis at pH 8compared to SEQ ID NO: 2 are shown below. L227M/L264R,A018K/L075D/T189D, A018K/P029E/T189D, D027S/N033D/T189D,G023K/V187N/L264R, L075R/D130A/V187T, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, D027E/L227M/N233Q/L264R,D027S/N033D/L075D/T189D, D027S/P029E/E045F/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/L075R/D130A/L264R, P029E/N073R/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027E/D130A/G163P/N233Q/L264R,D027S/L075R/V154I/T189Q/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/N094R/G156W/V187N/T189Q, K024A/L075R/V154I/G156W/V187Q,A018K/D027S/P029E/N033D/E045F/L075D,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-13 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 for p-nitrophenyl palmitate hydrolysis at pH 8 are shown below.A018K/E045F, D027S/N033D, D130A/L264R, D130A/V187T, G023K/L264R,K024A/L075Q, L075R/L264R, S058M/L264R, V187T/L264R, A018K/A049V/L075Q,A018K/D027S/E045F, A018K/E045F/T189D, A018K/L075D/T189D,A018K/L075Q/V187T, A018K/N033D/T189D, A018K/N073R/L075D,A018K/P029E/T189D, A049V/V187T/T189D, D027N/N233Q/P256T,D027Q/N233Q/P256T, D027S/N033D/T189D, D111A/D130A/L264R,D130A/V187N/L264R, F051T/I252Q/L264R, G023K/D130A/L264R,G023K/E056K/V187T, G023K/L075R/L264R, G023K/V187N/L264R,G023K/V187T/L264R, G023Q/L075Q/V187T, G163P/L227M/L264R,K024A/L075Q/V077I, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/G156W/V187N, L075Q/V187N/L264R, L075Q/V187T/L264R,L075R/D130A/L264R, L075R/D130A/V187T, P029E/N073R/L075D,Q004D/D027S/P256T, V187N/T189Q/L264R, A018K/D027S/N033D/L075D,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/V187T,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/N073R/L075D/T189D, A018K/P029E/N073R/L075D,D027E/D048Q/G163P/L264R, D027E/D130A/N233Q/L264R,D027E/D137Q/G163P/L227M, D027E/G163P/L227M/L264R,D027Q/S058M/I090F/P256T, D027S/D130A/I252Q/L264R,D027S/E045F/N073R/L075D, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, E056K/D130A/V187N/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,F051T/L075Q/I252Q/L264R, G023K/D027Q/F051T/L075Q,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/E056K/L075R/L264R, G023K/L075Q/D130A/V187N,G023K/L075Q/G156W/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/E045F/A049V/T189D,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/K024A/L075R/V154I, G023Q/L075Q/D130A/L264R,G023Q/L075Q/V187T/L264R, G091Q/V187T/I252Q/L264R,K024A/D130A/V154I/V187T, K024A/L075Q/D130A/G156W,K024A/L075R/G156W/V187N, L075G/D130A/V187T/I252Q,L075G/V187H/I252Q/L264R, L075Q/D130A/G156W/V187N,L075Q/V077I/D130A/V187Q, N011K/S058M/N233Q/P256T,P029E/E045F/N073R/T189D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/N011K/D027S/P256T, S058M/G163P/L227M/L264R,S058M/G163P/N233Q/L264R, S058M/L075Q/I252Q/L264R,A018K/D027S/N033D/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D130A/G156W/V187N/L264R, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/E045F/A049V/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/E045F/V077I/T189D, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/G156W, A018K/K024A/L075R/N094R/D130A,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,A018K/L075Q/N094R/G156W/V187N, A018K/L075Q/V077I/N094R/G156W,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/G163P/L227M/L264R,D027S/E056K/S058M/N233Q/P256T, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D027S/N033D/E045F/N073R/L075D,D027S/N033D/N073R/L075D/T189D, D027S/P029E/N073R/L075D/T189D,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/D137Q/N233Q/L264R, D048Q/S058M/G163P/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/D137Q/G163P/N233Q/L264R,E056K/L075G/V187N/I252Q/L264R, E056K/L075Q/G156W/T189Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/E056K/V187N/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187N, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/G156W/V187N,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,L075Q/V154I/V187N/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,N011K/G023K/D111A/G156W/L264R, N011K/G023K/E056K/L075Q/T189Q,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D130A/V187N/T189Q/L264R,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/V154I/V187T/T189Q/L264R,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,N011K/E056K/L075Q/D130A/V187N/T189Q,Q004D/D027N/E056K/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-14 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 for p-nitrophenyl palmitate hydrolysis at pH 8compared to TLL SEQ ID NO: 1 are shown below. A018K/E045F, D027S/N033D,D130A/L264R, D130A/V187T, G023K/L264R, L075R/L264R, S058M/L264R,V187T/L264R, A018K/L075D/T189D, A018K/N033D/T189D, A018K/P029E/T189D,D027S/N033D/T189D, D111A/D130A/L264R, D130A/V187N/L264R,F051T/I252Q/L264R, G023K/D130A/L264R, G023K/E056K/V187T,G023K/L075R/L264R, G023K/V187N/L264R, G023K/V187T/L264R,G163P/L227M/L264R, L075G/D130A/V187H, L075Q/V187N/L264R,L075Q/V187T/L264R, L075R/D130A/L264R, L075R/D130A/V187T,Q004D/D027S/P256T, V187N/T189Q/L264R, A018K/E045F/L075D/T189D,A018K/G023K/D111A/T189Q, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/G163P/L227M/L264R, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/V187N/L264R,G023K/D130A/V187T/L264R, G023K/E056K/L075R/L264R,G023K/L075R/D130A/L264R, G023K/L075R/V187T/L264R,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/N011K/D027S/P256T, S058M/G163P/L227M/L264R,S058M/G163P/N233Q/L264R, S058M/L075Q/I252Q/L264R,A018K/D027S/P029E/N033D/L075D, A018K/D130A/G156W/V187N/L264R,A018K/G023Q/K024A/L075Q/G156W, A018K/P029E/N033D/N073R/L075D,D027E/D048Q/D137Q/L227M/L264R, D027E/D048Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D130A/G163P/L264R,D027E/S058M/G163P/L227M/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/D137Q/N233Q/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075G/V187N/I252Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/E056K/V187N/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,N011K/G023K/D111A/G156W/L264R, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-15 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value for for p-nitrophenylpalmitate hydrolysis at pH 8 compared to TLL SEQ ID NO: 1 are shownbelow. D130A/L264R, D130A/V187T, A018K/L075D/T189D, A018K/P029E/T189D,D027S/N033D/T189D, G023K/V187N/L264R, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,Q004D/D027S/P256T, A018K/L075Q/V077I/N094R, D027S/D130A/I252Q/L264R,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,F051T/L075Q/I252Q/L264R, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/F051T/I252Q/L264R,G023Q/G091Q/I252Q/L264R, G091Q/V187T/I252Q/L264R,L075G/V187H/I252Q/L264R, P029E/N073R/L075D/T189D,Q004D/N011K/D027S/P256T, S058M/G163P/L227M/L264R,S058M/L075Q/I252Q/L264R, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/K024A/L075Q/G156W, A018K/P029E/N033D/N073R/L075D,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/D137Q/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/G091Q/I252Q/L264R,K024A/L075R/V154I/G156W/V187Q, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023Q/L075Q/G091Q/V187N/I252Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TALBE 3-16 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 for p-nitrophenyl palmitate hydrolysis at pH 8 areshown below. A018K/E045F, D027S/N033D, D111A/L264R, D130A/L264R,D130A/V187T, G023K/L264R, K024A/L075Q, L075R/L264R, S058M/L264R,V187T/L264R, A018K/A049V/L075Q, A018K/D027S/E045F, A018K/E045F/T189D,A018K/L075D/T189D, A018K/L075Q/V187T, A018K/N033D/T189D,A018K/N073R/L075D, A018K/P029E/T189D, A049V/V187T/T189D,D027N/N233Q/P256T, D027Q/N233Q/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D111A/D130A/L264R, D130A/V187N/L264R,E045F/L075D/T189D, F051T/I252Q/L264R, G023K/D130A/L264R,G023K/E056K/V187T, G023K/L075R/L264R, G023K/V187N/L264R,G023K/V187T/L264R, G023Q/E056K/L075R, G023Q/L075Q/V187T,G163P/L227M/L264R, K024A/L075Q/V077I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/G156W/V187N, L075Q/V187N/L264R,L075Q/V187T/L264R, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187T/L264R, N073R/L075D/T189D, P029E/N073R/L075D,Q004D/D027S/P256T, V187N/T189Q/L264R, A018K/D027S/N033D/L075D,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/L075Q/N094R/V187Q,A018K/L075Q/V077I/N094R, A018K/N073R/L075D/T189D,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/G163P/L227M/L264R, D027E/S058M/G163P/L264R,D027Q/S058M/I090F/P256T, D027S/D130A/I252Q/L264R,D027S/E045F/N073R/L075D, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, D111A/D130A/V154I/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/V187N/L264R,G023K/D130A/V187T/L264R, G023K/E056K/L075R/L264R,G023K/L075Q/D130A/V187N, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023K/L075R/V187T/L264R,G023Q/E045F/A049V/T189D, G023Q/F051T/I252Q/L264R,G023Q/G091Q/I252Q/L264R, G023Q/K024A/L075R/V154I,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, K024A/L075R/G156W/V187N,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,L075Q/D130A/G156W/V187N, L075Q/V077I/D130A/V187Q,N011K/S058M/N233Q/P256T, P029E/E045F/N073R/T189D,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/N011K/D027S/P256T,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/I252Q/L264R,A018K/D027S/N033D/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D130A/G156W/V187N/L264R, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/A049V/L075Q/V077I, A018K/G023Q/E045F/A049V/V187T,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/E045F/V077I/T189D,A018K/G023Q/K024A/L075Q/G156W, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/G156W, A018K/K024A/L075R/N094R/D130A,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,A018K/L075Q/N094R/G156W/V187N, A018K/L075Q/V077I/N094R/G156W,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D027S/E056K/S058M/N233Q/P256T,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075R/V154I/T189Q/L264R,D027S/N033D/E045F/N073R/L075D, D027S/N033D/N073R/L075D/T189D,D027S/P029E/N073R/L075D/T189D, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/D137Q/N233Q/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075G/V187N/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/E056K/V187N/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187N, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/G156W/V187N,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,L075Q/V154I/V187N/T189Q/L264R, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, N011K/G023K/D111A/G156W/L264R,N011K/G023K/E056K/L075Q/T189Q, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/V154I/V187T/T189Q/L264R,K024A/L075R/D111A/V154I/G156W/V187N,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,N011K/E056K/L075Q/D130A/V187N/T189Q,Q004D/D027N/E056K/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/L075Q/D130A/G156W/V187N/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-17 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 for p-nitrophenylpalmitate hydrolysis at pH 8 compared to SEQ ID NO: 2 are shown below.A018K/E045F, D027S/N033D, D111A/L264R, D130A/L264R, D130A/V187T,G023K/L264R, L075R/L264R, S058M/L264R, V187T/L264R, A018K/L075D/T189D,A018K/N033D/T189D, A018K/P029E/T189D, D027S/L075Q/G091Q,D027S/N033D/T189D, D111A/D130A/L264R, D130A/V187N/L264R,F051T/I252Q/L264R, G023K/D130A/L264R, G023K/E056K/V187T,G023K/L075R/L264R, G023K/V187N/L264R, G023K/V187T/L264R,G163P/L227M/L264R, L075G/D130A/V187H, L075Q/V187N/L264R,L075Q/V187T/L264R, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187T/L264R, Q004D/D027S/P256T, V187N/T189Q/L264R,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/L075Q/V077I/N094R, A018K/P029E/N073R/L075D,D027E/D048Q/G163P/L264R, D027E/D130A/N233Q/L264R,D027E/D137Q/G163P/L227M, D027E/G163P/L227M/L264R,D027E/S058M/G163P/L264R, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/V187N/L264R,G023K/D130A/V187T/L264R, G023K/E056K/L075R/L264R,G023K/L075R/D130A/L264R, G023K/L075R/V187T/L264R,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/N011K/D027S/P256T, S058M/G163P/L227M/L264R,S058M/G163P/N233Q/L264R, S058M/L075Q/G091Q/I252Q,S058M/L075Q/I252Q/L264R, A018K/D027S/P029E/N033D/L075D,A018K/D130A/G156W/V187N/L264R, A018K/G023Q/K024A/L075Q/G156W,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/D137Q/N233Q/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075G/V187N/I252Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075G/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/L264R, F051T/L075Q/V187N/I252Q/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/E056K/V187N/I252Q/L264R,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, K024A/L075R/V154I/G156W/V187Q,L075G/D130A/V187T/I252Q/L264R, N011K/G023K/D111A/G156W/L264R,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-18 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value for for p-nitrophenyl palmitate hydrolysis at pH 8 compared toSEQ ID NO: 2 are shown below. D130A/L264R, D130A/V187T,A018K/L075D/T189D, A018K/P029E/T189D, D027S/N033D/T189D,G023K/V187N/L264R, L075G/D130A/V187H, L075Q/V187N/L264R,L075R/D130A/L264R, L075R/D130A/V187T, Q004D/D027S/P256T,A018K/L075Q/V077I/N094R, D027S/D130A/I252Q/L264R,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,F051T/L075Q/I252Q/L264R, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/F051T/I252Q/L264R,G023Q/G091Q/I252Q/L264R, G091Q/V187T/I252Q/L264R,L075G/V187H/I252Q/L264R, P029E/N073R/L075D/T189D,Q004D/N011K/D027S/P256T, S058M/G163P/L227M/L264R,S058M/L075Q/I252Q/L264R, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/K024A/L075Q/G156W, A018K/P029E/N033D/N073R/L075D,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/D137Q/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/G091Q/I252Q/L264R,K024A/L075R/V154I/G156W/V187Q, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023Q/L075Q/G091Q/V187N/I252Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 3-19 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 for p- nitrophenyl caprylate (octanoate) hydrolysis at pH 6 areshown below. D027E/G163P, K024A/L075Q, P029E/N033D, A018K/D027S/E045F,A018K/L075D/T189D, A018K/L075Q/V187T, A018K/N073R/L075D,A018K/P029E/T189D, A049V/V187T/T189D, D027S/N033D/T189D,G023K/V187N/L264R, G023Q/L075Q/V187T, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/G156W/T189D, L075Q/G156W/V187N,L075R/D130A/L264R, L075R/D130A/V187T, N073R/L075D/T189D,P029E/N033D/E045F, P029E/N033D/L075D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/G023Q/A049V/T189D,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/V187T,A018K/L075Q/V077I/N094R, A018K/P029E/N033D/T189D,D027E/D137Q/L227M/L264R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D027Q/F051T/L075Q, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023Q/E045F/A049V/T189D,G023Q/K024A/L075R/V154I, K024A/L075Q/D130A/V154I,K024A/L075R/G156W/V187N, L075Q/D130A/G156W/V187N,L075Q/V077I/G156W/V187N, L075Q/V077I/V187T/T189D,P029E/E045F/L075D/T189D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, A018K/D027S/N033D/L075D/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D027S/P029E/N073R/L075D, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/E045F/A049V/L075Q, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/G156W/V187N, A018K/P029E/N033D/N073R/L075D,D027E/D048Q/S058M/G163P/N233Q, D027S/L075R/V154I/T189Q/L264R,D130A/D137Q/G163P/N233Q/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/V077I/D130A/G156W,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/D027S/V154I/V187N/L264R, G023Q/K024A/L075Q/G156W/V187Q,K024A/L075R/V154I/G156W/V187Q, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, N011K/D027N/E056K/N233Q/P256T,N011K/G023K/E056K/L075Q/T189Q, A018K/D027S/P029E/N033D/E045F/L075D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/E045F/A049V/L075Q/V187T,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/E056K/S058M/I090F/N233Q/P256T,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/P029E/N033D/E045F/N073R/T189D,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/V154I/V187T/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R

TABLE 3-20 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 for p-nitrophenyl caprylate (octanoate) hydrolysisat pH 6 compared to TLL SEQ ID NO: 1 are shown below. D027E/G163P,P029E/N033D, A018K/L075D/T189D, A018K/P029E/T189D, D027S/N033D/T189D,G023K/V187N/L264R, L075G/D130A/V187H, L075R/D130A/L264R,L075R/D130A/V187T, P029E/N033D/E045F, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, D027E/D137Q/L227M/L264R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, G023K/D027Q/F051T/L075Q,G023K/L075R/D130A/L264R, P029E/N073R/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027S/L075R/V154I/T189Q/L264R,D130A/D137Q/G163P/N233Q/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/N094R/G156W/V187N/T189Q, G023Q/K024A/L075Q/G156W/V187Q,K024A/L075R/V154I/G156W/V187Q, A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,G023K/K024A/L075Q/V154I/G156W/V187Q,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H

TABLE 3-21 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value for p-nitrophenylcaprylate (octanoate) hydrolysis at pH 6 compared to TLL SEQ ID NO: 1are shown below. A018K/P029E/T189D, D027S/N033D/T189D,G023K/V187N/L264R, L075R/D130A/V187T, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, D027S/P029E/E045F/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,P029E/N073R/L075D/T189D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/L075R/D130A/V154I, G023K/E056K/D130A/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, K024A/L075R/V154I/G156W/V187Q,A018K/D027S/P029E/N033D/E045F/L075D,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W

TABLE 3-22 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 for p-nitrophenyl caprylate (octanoate) hydrolysisat pH 6 are shown below. D027E/G163P, K024A/L075Q, A018K/D027S/E045F,A018K/L075D/T189D, A018K/L075Q/V187T, A018K/N073R/L075D,A018K/P029E/T189D, A049V/V187T/T189D, D027S/N033D/T189D,G023K/V187N/L264R, G023Q/L075Q/V187T, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/G156W/T189D, L075Q/G156W/V187N,L075R/D130A/L264R, L075R/D130A/V187T, N073R/L075D/T189D,P029E/N033D/L075D, A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/G023Q/A049V/T189D, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/V187T, A018K/L075Q/V077I/N094R,A018K/P029E/N033D/T189D, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D027Q/F051T/L075Q, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023Q/E045F/A049V/T189D,G023Q/K024A/L075R/V154I, K024A/L075Q/D130A/V154I,K024A/L075R/G156W/V187N, L075Q/D130A/G156W/V187N,L075Q/V077I/G156W/V187N, L075Q/V077I/V187T/T189D,P029E/E045F/L075D/T189D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, A018K/D027S/N033D/L075D/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D027S/P029E/N073R/L075D, A018K/G023K/K024A/L075R/N094R,A018K/G023Q/E045F/A049V/L075Q, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/G156W/V187N, A018K/P029E/N033D/N073R/L075D,D027E/D048Q/S058M/G163P/N233Q, D027S/L075R/V154I/T189Q/L264R,D130A/D137Q/G163P/N233Q/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/V077I/D130A/G156W,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/K024A/L075Q/G156W/V187Q, K024A/L075R/V154I/G156W/V187Q,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,N011K/D027N/E056K/N233Q/P256T, N011K/G023K/E056K/L075Q/T189Q,A018K/D027S/P029E/N033D/E045F/L075D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/E045F/A049V/L075Q/V187T,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/E056K/S058M/I090F/N233Q/P256T,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/P029E/N033D/E045F/N073R/T189D,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/V154I/V187T/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R

TABLE 3-23 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 for p-nitrophenylcaprylate (octanoate) hydrolysis at pH 6 compared to SEQ ID NO: 2 areshown below. D027E/G163P, A018K/L075D/T189D, A018K/P029E/T189D,D027S/N033D/T189D, G023K/V187N/L264R, L075G/D130A/V187H,L075R/D130A/L264R, L075R/D130A/V187T, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D027Q/F051T/L075Q, G023K/L075R/D130A/L264R,P029E/N073R/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027S/L075R/V154I/T189Q/L264R, D130A/D137Q/G163P/N233Q/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/N094R/G156W/V187N/T189Q,G023Q/K024A/L075Q/G156W/V187Q, K024A/L075R/V154I/G156W/V187Q,A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,G023K/K024A/L075Q/V154I/G156W/V187Q,K024A/L075Q/D111A/V154I/V187N/T189Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H

TABLE 3-24 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value for p-nitrophenyl caprylate (octanoate) hydrolysis at pH 6compared to SEQ ID NO: 2 are shown below. A018K/P029E/T189D,D027S/N033D/T189D, G023K/V187N/L264R, L075R/D130A/V187T,A018K/D027S/P029E/T189D, A018K/L075Q/V077I/N094R,D027S/P029E/E045F/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, P029E/N073R/L075D/T189D,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,G023K/E056K/D130A/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, A018K/D027S/P029E/N033D/E045F/L075D,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W

TABLE 3-25 TLL variants with increased pNPP/pNPB or pNPO/pNPB specificactivity ratio compared to TLL SEQ ID NO: 1 are shown below.A018K/E045F, D111A/L264R, D130A/V187T, G023K/L264R, L075R/L264R,S058M/L264R, V187T/L264R, A018K/E045F/T189D, A018K/G023K/L075Q,A018K/N033D/T189D, D027N/N233Q/P256T, D027Q/N233Q/P256T,D027Q/S058M/P256T, D111A/D130A/L264R, D130A/V154I/G156W,D130A/V187N/L264R, F051T/I252Q/L264R, G023K/D130A/L264R,G023K/E056K/V187T, G023K/L075R/L264R, G023K/V187T/L264R,G163P/L227M/L264R, L075Q/V187T/L264R, Q004D/D027S/P256T,A018K/D130A/G156W/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/V077I/G156W,D027E/D048Q/G163P/L264R, D027E/D130A/N233Q/L264R,D027E/G163P/L227M/L264R, D027S/D130A/I252Q/L264R,D027S/E045F/N073R/L075D, D027S/N033D/E045F/T189D,D048Q/D130A/G163P/L264R, D111A/D130A/V154I/G156W,D111A/D130A/V187T/L264R, E056K/D130A/V187N/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,F051T/L075Q/I252Q/L264R, G023K/D130A/V187N/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/L075R/V187T/L264R, G023Q/E045F/A049V/V187T,G023Q/E045F/L075Q/G156W, G023Q/F051T/I252Q/L264R,G023Q/G091Q/I252Q/L264R, G023Q/L075Q/D130A/L264R,G023Q/L075Q/V187T/L264R, G091Q/V187T/I252Q/L264R,K024A/D130A/V154I/V187T, L075G/D130A/V187T/I252Q,L075G/V187H/I252Q/L264R, N011K/S058M/N233Q/P256T,P029E/E045F/N073R/T189D, Q004D/D027N/E056K/P256T,Q004D/N011K/D027N/P256T, Q004D/N011K/D027S/P256T,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/I252Q/L264R, A018K/D130A/G156W/V187N/L264R,A018K/E045F/A049V/L075Q/T189D, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/A049V/L075Q/V187T,A018K/G023Q/E045F/V077I/T189D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/N094R/G156W/V187T, A018K/L075Q/N094R/D130A/V187N,D027E/D048Q/D137Q/L227M/L264R, D027E/D048Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D130A/G163P/L264R,D027N/E056K/S058M/N233Q/P256T, D027S/E056K/S058M/N233Q/P256T,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075R/V154I/T189Q/L264R,D027S/N033D/E045F/N073R/L075D, D027S/P029E/N033D/E045F/N073R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/L075G/V187N/I252Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/E056K/D130A/V187N/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/E045F/A049V/V077I/T189D, G023Q/E045F/L075Q/V077I/V187T,G023Q/E056K/V187N/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, K024A/L075R/V154I/G156W/V187Q,L075G/D130A/V187T/I252Q/L264R, L075Q/D111A/D130A/V187T/T189Q,L075Q/V077I/D130A/G156W/V187N, N011K/D027N/S058M/N233Q/P256T,N011K/D027Q/S058M/N233Q/P256T, S058M/D130A/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156W/V187T,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/D130A/G156W/T189Q/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,Q004D/D027N/E056K/S058M/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027S/I090F/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/G023Q/E045F/A049V/L075Q/G156W/V187T,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,A018K/L075Q/D130A/G156W/V187N/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-26 TLL variants with Performance Index for expression ≥0.5 andincreased pNPP/pNPB or pNPO/pNPB specific activity ratio compared to TLLSEQ ID NO: 1 are shown below. A018K/E045F, D111A/L264R, D130A/V187T,G023K/L264R, L075R/L264R, S058M/L264R, V187T/L264R, A018K/N033D/T189D,D027Q/S058M/P256T, D111A/D130A/L264R, D130A/V154I/G156W,D130A/V187N/L264R, F051T/I252Q/L264R, G023K/D130A/L264R,G023K/E056K/V187T, G023K/L075R/L264R, G023K/V187T/L264R,G163P/L227M/L264R, L075Q/V187T/L264R, Q004D/D027S/P256T,A018K/D130A/G156W/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/V077I/G156W,D027E/D048Q/G163P/L264R, D027E/D130A/N233Q/L264R,D027E/G163P/L227M/L264R, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D048Q/D130A/G163P/L264R,D111A/D130A/V154I/G156W, D111A/D130A/V187T/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/L075R/L264R, G023K/L075R/V187T/L264R,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,Q004D/D027N/E056K/P256T, Q004D/N011K/D027S/P256T,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/I252Q/L264R, A018K/D130A/G156W/V187N/L264R,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/N094R/G156W/V187T,D027E/D048Q/D137Q/L227M/L264R, D027E/D048Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D130A/G163P/L264R,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075R/V154I/T189Q/L264R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/L075G/V187N/I252Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/E056K/D130A/V187N/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/E056K/V187N/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, K024A/L075R/V154I/G156W/V187Q,L075G/D130A/V187T/I252Q/L264R, N011K/D027N/S058M/N233Q/P256T,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,Q004D/N011K/D027N/I090F/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-27 TLL variants with Performance Index for expression ≥0.5 andpNPP/pNPB or pNPO/pNPB ≥50% of the maximum specific activity ratiocompared to TLL SEQ ID NO: 1 are shown below. D130A/V187T,D027Q/S058M/P256T, F051T/I252Q/L264R, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187T/T189Q, G023K/L075R/V187T/L264R,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/V187T/L264R, G091Q/V187T/I252Q/L264R,L075G/V187H/I252Q/L264R, Q004D/N011K/D027S/P256T,S058M/G163P/L227M/L264R, S058M/L075Q/I252Q/L264R,D027E/D048Q/D137Q/L227M/L264R, D027E/D048Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D048Q/D137Q/G163P/N233Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/E056K/D130A/V187N/L264R, G023K/K024A/D111A/G156W/T189Q,G023Q/E056K/V187N/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,N011K/D027N/S058M/N233Q/P256T, A018K/G023Q/K024A/V077I/V154I/V187N,A018K/K024A/D111A/V187N/T189Q/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,A018K/K024A/D130A/V154I/G156W/V187T/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R

TABLE 3-28 TLL variants with increased pNPP/pNPB or pNPO/pNPB specificactivity ratio compared to Reference sequence SEQ ID NO: 2 are shownbelow. A018K/E045F, D130A/V187T, G023K/L264R, S058M/L264R,A018K/E045F/T189D, A018K/G023K/L075Q, D027N/N233Q/P256T,D027Q/N233Q/P256T, D130A/V187N/L264R, F051T/I252Q/L264R,G023K/D130A/L264R, G023K/V187T/L264R, Q004D/D027S/P256T,D027E/D130A/N233Q/L264R, D027E/G163P/L227M/L264R,D027S/D130A/I252Q/L264R, D027S/E045F/N073R/L075D,D048Q/D130A/G163P/L264R, E056K/D130A/V187N/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,F051T/L075Q/I252Q/L264R, G023K/D130A/V187N/L264R,G023K/E056K/L075R/L264R, G023K/L075R/V187T/L264R,G023Q/E045F/A049V/V187T, G023Q/E045F/L075Q/G156W,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/V187T/L264R, G091Q/V187T/I252Q/L264R,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,N011K/S058M/N233Q/P256T, P029E/E045F/N073R/T189D,Q004D/N011K/D027S/P256T, S058M/G163P/L227M/L264R,S058M/L075Q/I252Q/L264R, A018K/D130A/G156W/V187N/L264R,A018K/G023Q/E045F/V077I/T189D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/G163P/L227M/N233Q/L264R,D027S/E056K/S058M/N233Q/P256T, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/N033D/E045F/N073R/L075D, D027S/P029E/N033D/E045F/N073R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, E056K/L075G/V187N/I252Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/E056K/D130A/V187N/L264R, G023K/L075R/D130A/V187N/L264R,G023Q/E045F/A049V/V077I/T189D, G023Q/E045F/L075Q/V077I/V187T,G023Q/E056K/V187N/I252Q/L264R, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,L075G/D130A/V187T/I252Q/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,Q004D/D027N/E056K/S058M/N233Q/P256T,Q004D/N011K/D027S/I090F/N233Q/P256T,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R

TABLE 3-29 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and increased pNPP/pNPB or pNPO/pNPBspecific activity ratio compared to SEQ ID NO: 2 are shown below.A018K/E045F, D130A/V187T, G023K/L264R, S058M/L264R, D130A/V187N/L264R,F051T/I252Q/L264R, G023K/D130A/L264R, G023K/V187T/L264R,Q004D/D027S/P256T, D027E/D130A/N233Q/L264R, D027E/G163P/L227M/L264R,D027S/D130A/I252Q/L264R, D048Q/D130A/G163P/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/E056K/L075R/L264R,G023K/L075R/V187T/L264R, G023Q/F051T/I252Q/L264R,G023Q/G091Q/I252Q/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, L075G/D130A/V187T/I252Q,L075G/V187H/I252Q/L264R, Q004D/N011K/D027S/P256T,S058M/G163P/L227M/L264R, S058M/L075Q/I252Q/L264R,A018K/D130A/G156W/V187N/L264R, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/G163P/L227M/N233Q/L264R,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D137Q/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,E056K/L075G/V187N/I252Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/V187N/I252Q/L264R,F051T/L075Q/V187N/I252Q/L264R, G023K/E056K/D130A/V187N/L264R,G023K/L075R/D130A/V187N/L264R, G023Q/E056K/V187N/I252Q/L264R,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, L075G/D130A/V187T/I252Q/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R

TABLE 3-30 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and pNPP/pNPB or pNPO/pNPB ≥50% of themaximum specific activity ratio compared to SEQ ID NO: 2 are shownbelow. D130A/V187T, F051T/I252Q/L264R, D027S/D130A/I252Q/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,F051T/L075Q/I252Q/L264R, G023K/L075R/V187T/L264R,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/V187T/L264R, G091Q/V187T/I252Q/L264R,L075G/V187H/I252Q/L264R, Q004D/N011K/D027S/P256T,S058M/G163P/L227M/L264R, S058M/L075Q/I252Q/L264R,D027E/D048Q/D137Q/L227M/L264R, D027E/D048Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L264R, D027E/D130A/G163P/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D048Q/D137Q/G163P/N233Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/E056K/D130A/V187N/L264R, G023Q/E056K/V187N/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, A018K/K024A/D111A/V187N/T189Q/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,A018K/K024A/D130A/V154I/G156W/V187T/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R

TABLE 3-31 TLL variants with increased pNPB/pNPP specific activity ratiocompared to TLL SEQ ID NO: 1 are shown below. A018K/L075D, A018K/T189D,D027N/E056K, D027N/N233Q, D027S/N033D, D027S/P256T, D130A/L264R,D130A/V187N, E045F/N073R, K024A/L075Q, K024A/V154I, L075Q/D130A,N094R/G156W, P029E/N033D, A018K/A049V/V187T, A018K/E045F/N073R,A018K/E045F/V187T, A018K/G023K/G156W, A018K/K024A/V154I,A018K/L075D/T189D, A049V/V187T/T189D, D027E/S058M/G163P,D027N/S058M/P256T, D027S/L075Q/G091Q, D027S/N033D/T189D,D027S/S058M/P256T, D130A/V187T/L264R, E045F/L075D/T189D,E056K/D130A/L264R, G023K/D130A/V187T, G023Q/A049V/T189D,G023Q/D130A/G156W, G023Q/K024A/G156W, G023Q/L075Q/V187T,K024A/D130A/V154I, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/G156W/V187N, L075Q/V187N/L264R, L075R/D130A/L264R,L075R/D130A/V187T, L075R/V187T/L264R, N011K/D027S/S058M,N011K/N233Q/P256T, N073R/L075D/T189D, N094R/D130A/V187T,P029E/N033D/E045F, V187N/T189Q/L264R, A018K/D027S/N033D/L075D,A018K/D111A/G156W/T189Q, A018K/E045F/A049V/G156W,A018K/E045F/A049V/V187T, A018K/E045F/L075D/T189D,A018K/E045F/L075Q/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/V187T, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D130A, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, A018K/V154I/G156W/V187T,D027E/D137Q/G163P/L227M, D027E/S058M/G163P/L264R,D027N/E056K/N233Q/P256T, D027N/E056K/S058M/P256T,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/T189D,D027S/E056K/D111A/V154I, D027S/E056K/I090F/P256T,D027S/N033D/E045F/N073R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/L075D, D027S/P029E/E045F/N073R,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/G163P/N233Q/L264R, D111A/D130A/V154I/G156W,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,E056K/D130A/V187T/L264R, G023K/D027Q/F051T/L075Q,G023K/D130A/G156W/T189Q, G023K/K024A/V154I/V187N,G023K/L075Q/G156W/V187N, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, G023Q/D027S/D111A/G156W,G023Q/K024A/D130A/G156W, G023Q/K024A/L075R/V154I,G023Q/K024A/V077I/G156W, G023Q/V154I/G156W/V187N,K024A/L075R/G156W/V187N, L075G/G091Q/V187N/L264R,L075Q/D130A/G156W/V187N, L075Q/V077I/G156W/V187N,L075Q/V187N/T189Q/L264R, L075R/D130A/V187T/L264R,N011K/D027N/E056K/S058M, N011K/D027Q/S058M/P256T,N011K/D027S/E056K/P256T, N011K/D027S/N233Q/P256T,N011K/E056K/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/E045F/N073R/L075D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, Q004D/D027N/S058M/P256T,Q004D/D027S/I090F/P256T, S058M/D137Q/G163P/N233Q,S058M/L075Q/G091Q/I252Q, A018K/A049V/L075Q/V187T/T189D,A018K/D027S/E045F/L075D/T189D, A018K/D027S/N033D/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/E045F/A049V/L075Q/V187T,A018K/G023K/D130A/V154I/G156W, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/L075R/N094R, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V187T, A018K/G023Q/E045F/A049V/V187T,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/D130A/G156W,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/T189D, A018K/K024A/N094R/D130A/V187N,A018K/K024A/N094R/G156W/V187T, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/G156W/V187N, A018K/P029E/E045F/N073R/L075D,A018K/P029E/N033D/N073R/L075D, A018K/V077I/G156W/V187T/T189D,D027E/S058M/D137Q/G163P/N233Q, D027E/S058M/G163P/L227M/L264R,D027Q/E056K/I090F/N233Q/P256T, D027Q/E056K/S058M/N233Q/P256T,D027S/P029E/N033D/E045F/N073R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,D130A/G163P/L227M/N233Q/L264R, E045F/A049V/L075Q/V187T/T189D,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/V154I/T189Q,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/E045F/A049V/G156W/T189D, G023Q/E045F/A049V/L075Q/V077I,G023Q/K024A/L075Q/G156W/V187Q, G023Q/K024A/L075R/D130A/V154I,G023Q/K024A/L075R/V077I/D130A, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/D111A/V154I/V187T, K024A/L075R/G156W/V187N/T189Q,L075Q/D130A/V187T/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,N011K/D027N/S058M/N233Q/P256T, N011K/D027Q/E056K/S058M/N233Q,N011K/D027Q/S058M/N233Q/P256T, N011K/D027S/E056K/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,P029E/N033D/E045F/L075D/T189D, Q004D/D027N/E056K/S058M/P256T,Q004D/D027N/I090F/N233Q/P256T, Q004D/D027Q/E056K/S058M/P256T,Q004D/N011K/D027N/N233Q/P256T, S058M/D130A/D137Q/G163P/L264R,S058M/D137Q/G163P/L227M/L264R, V077I/D130A/V154I/G156W/V187N,A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/A049V/L075Q/V077I/G156W,A018K/G023Q/A049V/V077I/V187T/T189D,A018K/G023Q/E045F/A049V/G156W/V187T,A018K/G023Q/E045F/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156C/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D111A/V154I/G156W/V187N,A018K/L075Q/D111A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,A018K/P029E/N033D/E045F/L075D/T189D,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/P029E/N033D/E045F/N073R/L075D,D027S/P029E/N033D/E045F/N073R/T189D,D027S/P029E/N033D/N073R/L075D/T189D,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/N094R/D111A/G156W/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,K024A/L075Q/V154I/V187T/T189Q/L264R,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/N094R/D111A/G156W/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027N/E056K/I090F/N233Q/P256T,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/E045F/L075Q/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/N094R/G156W/V187N,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027S/E056K/L075R/D111A/G156W/V187N/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/N094R/D130A/V154I/V187N/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-32 TLL variants with Performance Index for expression ≥0.5 andincreased pNPB/pNPP specific activity ratio compared to TLL SEQ ID NO: 1are shown below. A018K/L075D, D027S/N033D, D027S/P256T, D130A/L264R,D130A/V187N, E045F/N073R, K024A/V154I, N094R/G156W, P029E/N033D,A018K/A049V/V187T, A018K/E045F/N073R, A018K/E045F/V187T,A018K/K024A/V154I, A018K/L075D/T189D, D027E/S058M/G163P,D027N/S058M/P256T, D027S/L075Q/G091Q, D027S/N033D/T189D,D027S/S058M/P256T, D130A/V187T/L264R, E056K/D130A/L264R,G023K/D130A/V187T, G023Q/A049V/T189D, G023Q/D130A/G156W,G023Q/K024A/G156W, K024A/D130A/V154I, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187T/L264R, N011K/N233Q/P256T, N094R/D130A/V187T,P029E/N033D/E045F, V187N/T189Q/L264R, A018K/D111A/G156W/T189Q,A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/V187T,A018K/L075Q/V077I/N094R, A018K/P029E/N073R/L075D,D027E/D137Q/G163P/L227M, D027E/S058M/G163P/L264R,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/N073R, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/G163P/N233Q/L264R,D111A/D130A/V154I/G156W, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, E056K/D130A/V187T/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, G023Q/K024A/D130A/G156W,G023Q/K024A/V077I/G156W, L075G/G091Q/V187N/L264R,L075R/D130A/V187T/L264R, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/N073R/L075D/T189D, Q004D/D027N/S058M/P256T,S058M/D137Q/G163P/N233Q, S058M/L075Q/G091Q/I252Q,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023K/D130A/V154I/G156W, A018K/G023K/K024A/D130A/G156W,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/V077I/D130A/G156W,A018K/K024A/N094R/D130A/V187N, A018K/K024A/N094R/G156W/V187T,A018K/P029E/N033D/N073R/L075D, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/N094R/G156W/V187N/T189Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075Q/V077I/D130A/G156W, N011K/D027N/S058M/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,Q004D/N011K/D027N/N233Q/P256T, S058M/D130A/D137Q/G163P/L264R,S058M/D137Q/G163P/L227M/L264R, V077I/D130A/V154I/G156W/V187N,A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 3-33 TLL variants with Performance Index for expression ≥0.5 andpNPB/pNPP ≥50% of the maximum specific activity ratio compared to TLLSEQ ID NO: are shown below. A018K/L075D, D027S/P256T, E045F/N073R,K024A/V154I, N094R/G156W, A018K/E045F/N073R, A018K/L075D/T189D,D027S/N033D/T189D, E056K/D130A/L264R, G023Q/A049V/T189D,K024A/D130A/V154I, L075R/D130A/L264R, L075R/D130A/V187T,N094R/D130A/V187T, P029E/N033D/E045F, A018K/E045F/A049V/G156W,A018K/E045F/L075D/T189D, A018K/P029E/N073R/L075D,D027Q/E056K/N233Q/P256T, D027S/E045F/L075D/T189D,D027S/E045F/N073R/T189D, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/P029E/E045F/N073R,D027S/P029E/N033D/L075D, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, G023K/D027Q/F051T/L075Q,G023K/L075R/D130A/L264R, G023Q/A049V/V077I/G156W,L075G/G091Q/V187N/L264R, N011K/D027S/N233Q/P256T,N033D/E045F/N073R/T189A, P029E/N073R/L075D/T189D,S058M/D137Q/G163P/N233Q, A018K/D027S/E045F/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/V077I/D130A/G156W,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,E045F/A049V/L075Q/V187T/T189D, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/N094R/G156W/V187N/T189Q, G023Q/L075Q/V077I/D130A/G156W,N011K/D027S/I090F/N233Q/P256T, Q004D/N011K/D027N/N233Q/P256T,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/V077I/N094R/D130A/V187N,Q004D/N011K/D027N/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 3-34 TLL variants with increased pNPB/pNPP specific activity ratiocompared to Reference sequence SEQ ID NO: 2 are shown below.A018K/L075D, D027N/E056K, D027S/N033D, D027S/P256T, D130A/V187N,E045F/N073R, K024A/L075Q, K024A/V154I, L075Q/D130A, N094R/G156W,P029E/N033D, A018K/E045F/N073R, A018K/E045F/V187T, A018K/G023K/G156W,A018K/K024A/V154I, A018K/L075D/T189D, A049V/V187T/T189D,D027E/S058M/G163P, D027S/L075Q/G091Q, D027S/N033D/T189D,D027S/S058M/P256T, D130A/V187T/L264R, E045F/L075D/T189D,E056K/D130A/L264R, G023K/D130A/V187T, G023Q/A049V/T189D,G023Q/K024A/G156W, G023Q/L075Q/V187T, K024A/D130A/V154I,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/G156W/V187N,L075R/D130A/L264R, L075R/D130A/V187T, L075R/V187T/L264R,N011K/D027S/S058M, N073R/L075D/T189D, N094R/D130A/V187T,P029E/N033D/E045F, A018K/D027S/N033D/L075D, A018K/D111A/G156W/T189Q,A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/E045F/L075Q/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/V187T, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D130A, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, A018K/V154I/G156W/V187T,D027E/D137Q/G163P/L227M, D027N/E056K/N233Q/P256T,D027N/E056K/S058M/P256T, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027S/E045F/L075D/T189D,D027S/E045F/N073R/T189D, D027S/E056K/D111A/V154I,D027S/E056K/I090F/P256T, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/L075D, D027S/P029E/E045F/N073R,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/K024A/V154I/V187N, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023Q/A049V/V077I/G156W,G023Q/D027S/D111A/G156W, G023Q/K024A/L075R/V154I,G023Q/V154I/G156W/V187N, K024A/L075R/G156W/V187N,L075G/G091Q/V187N/L264R, L075Q/D130A/G156W/V187N,L075Q/V077I/G156W/V187N, L075Q/V187N/T189Q/L264R,N011K/D027N/E056K/S058M, N011K/D027Q/S058M/P256T,N011K/D027S/E056K/P256T, N011K/D027S/N233Q/P256T,N011K/E056K/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/E045F/N073R/L075D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, Q004D/D027N/S058M/P256T,Q004D/D027S/I090F/P256T, S058M/D137Q/G163P/N233Q,A018K/A049V/L075Q/V187T/T189D, A018K/D027S/E045F/L075D/T189D,A018K/D027S/N033D/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/E045F/A049V/L075Q/V187T, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/L075R/N094R, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V187T, A018K/G023Q/E045F/A049V/V187T,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/D130A/G156W,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/T189D, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/G156W/V187N,A018K/P029E/E045F/N073R/L075D, A018K/P029E/N033D/N073R/L075D,A018K/V077I/G156W/V187T/T189D, D027E/S058M/D137Q/G163P/N233Q,D027Q/E056K/I090F/N233Q/P256T, D027Q/E056K/S058M/N233Q/P256T,D027S/P029E/N033D/E045F/N073R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/G163P/L227M/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/D130A/V154I/T189Q, G023K/N094R/G156W/V187N/T189Q,G023Q/A049V/L075Q/G156W/V187T, G023Q/E045F/A049V/G156W/T189D,G023Q/E045F/A049V/L075Q/V077I, G023Q/K024A/L075Q/G156W/V187Q,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/V077I/D130A,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/D111A/V154I/V187T,K024A/L075R/G156W/V187N/T189Q, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075R/D130A/V154I/T189Q/L264R,N011K/D027Q/E056K/S058M/N233Q, N011K/D027Q/S058M/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, P029E/N033D/E045F/L075D/T189D,Q004D/D027N/E056K/S058M/P256T, Q004D/D027N/I090F/N233Q/P256T,Q004D/D027Q/E056K/S058M/P256T, Q004D/N011K/D027N/N233Q/P256T,S058M/D130A/D137Q/G163P/L264R, S058M/D137Q/G163P/L227M/L264R,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/A049V/L075Q/V077I/G156W,A018K/G023Q/A049V/V077I/V187T/T189D,A018K/G023Q/E045F/A049V/G156W/V187T,A018K/G023Q/E045F/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156C/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D111A/V154I/G156W/V187N,A018K/L075Q/D111A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,A018K/P029E/N033D/E045F/L075D/T189D,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/P029E/N033D/E045F/N073R/L075D,D027S/P029E/N033D/E045F/N073R/T189D,D027S/P029E/N033D/N073R/L075D/T189D,D048Q/S058M/D130A/D137Q/G163P/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/N094R/D111A/G156W/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/N094R/D111A/G156W/T189Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/D027N/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/E045F/L075Q/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/N094R/G156W/V187N,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027S/E056K/L075R/D111A/G156W/V187N/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/N094R/D130A/V154I/V187N/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R

TABLE 3-35 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and increased pNPB/pNPP specific activityratio compared to Reference sequence SEQ ID NO: 2 are shown below.A018K/L075D, D027S/N033D, D027S/P256T, D130A/V187N, E045F/N073R,K024A/V154I, N094R/G156W, P029E/N033D, A018K/E045F/N073R,A018K/E045F/V187T, A018K/K024A/V154I, A018K/L075D/T189D,D027E/S058M/G163P, D027S/L075Q/G091Q, D027S/N033D/T189D,D027S/S058M/P256T, D130A/V187T/L264R, E056K/D130A/L264R,G023K/D130A/V187T, G023Q/A049V/T189D, G023Q/K024A/G156W,K024A/D130A/V154I, L075G/D130A/V187H, L075R/D130A/L264R,L075R/D130A/V187T, L075R/V187T/L264R, N094R/D130A/V187T,P029E/N033D/E045F, A018K/D111A/G156W/T189Q, A018K/E045F/A049V/G156W,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/L075Q/V077I/N094R,A018K/P029E/N073R/L075D, D027E/D137Q/G163P/L227M,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/N073R,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, L075G/G091Q/V187N/L264R,N011K/D027Q/S058M/P256T, N011K/D027S/N233Q/P256T,N033D/E045F/N073R/T189A, P029E/N073R/L075D/T189D,Q004D/D027N/S058M/P256T, S058M/D137Q/G163P/N233Q,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023K/D130A/V154I/G156W, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/V077I/D130A/G156W, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027E/S058M/D137Q/G163P/N233Q,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/G163P/L227M/L264R, D048Q/S058M/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/N094R/G156W/V187N/T189Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075Q/V077I/D130A/G156W, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, Q004D/N011K/D027N/N233Q/P256T,S058M/D130A/D137Q/G163P/L264R, S058M/D137Q/G163P/L227M/L264R,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 3-36 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and pNPB/pNPP ≥50% of the maximum specificactivity ratio compared to Reference sequence SEQ ID NO: 2 are shownbelow. A018K/L075D, D027S/P256T, E045F/N073R, K024A/V154I, N094R/G156W,A018K/E045F/N073R, A018K/L075D/T189D, D027S/N033D/T189D,E056K/D130A/L264R, G023Q/A049V/T189D, K024A/D130A/V154I,L075R/D130A/L264R, L075R/D130A/V187T, N094R/D130A/V187T,P029E/N033D/E045F, A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/P029E/N073R/L075D, D027Q/E056K/N233Q/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/L075D/T189D,D027S/P029E/E045F/N073R, D027S/P029E/N033D/L075D,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,G023K/D027Q/F051T/L075Q, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, L075G/G091Q/V187N/L264R,N011K/D027S/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/N073R/L075D/T189D, S058M/D137Q/G163P/N233Q,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/G023Q/V077I/D130A/G156W, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, E045F/A049V/L075Q/V187T/T189D,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/N094R/G156W/V187N/T189Q,G023Q/L075Q/V077I/D130A/G156W, N011K/D027S/I090F/N233Q/P256T,Q004D/N011K/D027N/N233Q/P256T, V077I/D130A/V154I/G156W/V187N,A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/V077I/N094R/D130A/V187N,Q004D/N011K/D027N/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

Example 4 Thermal Stability of TLL Combinatorial Variants

The thermal stability of TLL variants created as described in Example 2was assayed as described in Example 1 (Thermostability Assay). Theperformance index was calculated for the variants compared to TLL, SEQID NO:1 or the reference sequence SEQ ID NO: 2.

TABLE 4-1 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 in the Thermostability assay are shown below. A018K/D027S,A018K/L075D, A018K/L075Q, A018K/T189D, D027E/G163P, D027N/E056K,D027N/N233Q, D027S/N033D, D027S/P256T, D111A/L264R, D130A/L264R,D130A/T189Q, D130A/V187N, D130A/V187T, E045F/N073R, G023K/D130A,G023K/L264R, G023Q/L075Q, K024A/L075Q, K024A/V154I, L075Q/D130A,L075Q/G156W, L075R/L264R, L227M/L264R, N011K/D027S, N073R/L075D,N094R/G156W, P029E/N033D, S058M/L264R, V187T/L264R, A018K/A049V/L075Q,A018K/A049V/V187T, A018K/D027S/E045F, A018K/D027S/N073R,A018K/D027S/T189D, A018K/E045F/T189D, A018K/E045F/V187T,A018K/G023K/G156W, A018K/G023K/L075Q, A018K/G023Q/L075Q,A018K/K024A/V154I, A018K/L075D/T189D, A018K/L075Q/G156W,A018K/L075Q/V187T, A018K/N033D/L075D, A018K/N033D/T189D,A018K/N073R/L075D, A018K/P029E/T189D, A018K/V154I/G156W,A049V/V187T/T189D, D027E/S058M/G163P, D027N/N233Q/P256T,D027N/S058M/P256T, D027Q/N233Q/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/N073R/L075D,D027S/N233Q/P256T, D027S/S058M/P256T, D111A/D130A/L264R,D130A/T189Q/L264R, D130A/V154I/G156W, D130A/V187N/L264R,D130A/V187T/L264R, E045F/L075D/T189D, E045F/N073R/L075D,E056K/D130A/L264R, E056K/D130A/T189Q, G023K/D130A/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,G023Q/D111A/L264R, G023Q/D130A/G156W, G023Q/E056K/L075R,G023Q/K024A/G156W, G023Q/L075Q/V187T, G163P/L227M/L264R,K024A/D130A/V154I, K024A/L075Q/G156W, K024A/L075Q/V077I,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075Q/G156W/T189D, L075Q/G156W/V187N, L075Q/G156W/V187T,L075Q/N094R/V154I, L075Q/V077I/T189D, L075Q/V154I/V187T,L075Q/V187T/L264R, L075Q/V187T/T189D, L075R/D130A/L264R,L075R/D130A/V187T, L075R/V187N/L264R, L075R/V187T/L264R,N011K/D027S/S058M, N011K/N233Q/P256T, N033D/N073R/T189D,N073R/L075D/T189D, N094R/D130A/V187T, P029E/E045F/L075D,P029E/L075D/T189D, P029E/N033D/L075D, P029E/N073R/L075D,Q004D/D027S/P256T, Q004D/N233Q/P256T, S058M/L227M/L264R,V077I/D130A/V154I, V077I/V187A/T189D, V187N/T189Q/L264R,A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/N073R, A018K/D027S/E045F/T189D,A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/E045F/A049V/V187T,A018K/E045F/L075D/T189D, A018K/E045F/L075Q/V187T,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/T189D,A018K/G023Q/A049V/V187T, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/G156W/V187T,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V077I,A018K/G023Q/L075Q/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/G156W, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/K024A/D130A/G156W,A018K/L075Q/G156W/T189D, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D111A, A018K/L075Q/N094R/D130A,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/L075Q/V187T/T189D, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/L075D,A018K/P029E/N033D/T189D, A018K/P029E/N073R/L075D,A049V/L075Q/V187T/T189D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/G163P/L227M/L264R,D027E/L227M/N233Q/L264R, D027E/S058M/G163P/L264R,D027N/E056K/N233Q/P256T, D027N/E056K/S058M/P256T,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E056K/D111A/V154I, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/N033D/N073R/L075D,D027S/N033D/N073R/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D027S/P029E/N033D/T189D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, D111A/D130A/V154I/G156W,D111A/D130A/V154I/L264R, D130A/G163P/L227M/L264R,D130A/G163P/N233Q/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, E056K/D130A/V187N/L264R,E056K/D130A/V187T/L264R, E056K/L075Q/V187N/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D027S/L075Q/L264R,G023K/D111A/D130A/L264R, G023K/D130A/G156W/T189Q,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/D130A/L264R,G023K/E056K/L075R/L264R, 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A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,A018K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187T/L264R,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/E056K/L075Q/D111A/V154I/V187N/T189Q/L264R,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/D027S/L075R/V154I/G156W/V187N/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023K/L075Q/D111A/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/D130A/V187T/T189Q/L264K,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 4-2 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 in the Thermostability assay compared to TLL SEQ IDNO: 1 are shown below. A018K/L075D, D027E/G163P, D027S/N033D,D027S/P256T, D111A/L264R, D130A/L264R, D130A/V187N, D130A/V187T,E045F/N073R, G023K/L264R, K024A/V154I, L075R/L264R, L227M/L264R,N094R/G156W, P029E/N033D, S058M/L264R, V187T/L264R, A018K/A049V/V187T,A018K/E045F/V187T, A018K/K024A/V154I, A018K/L075D/T189D,A018K/N033D/T189D, A018K/P029E/T189D, A018K/V154I/G156W,D027E/S058M/G163P, D027N/S058M/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/S058M/P256T,D111A/D130A/L264R, D130A/V154I/G156W, D130A/V187N/L264R,D130A/V187T/L264R, E056K/D130A/L264R, G023K/D130A/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,G023Q/D130A/G156W, G023Q/K024A/G156W, G163P/L227M/L264R,K024A/D130A/V154I, L075G/D130A/V187H, L075Q/V187T/L264R,L075R/D130A/L264R, L075R/D130A/V187T, L075R/V187N/L264R,L075R/V187T/L264R, N011K/N233Q/P256T, N094R/D130A/V187T,Q004D/D027S/P256T, V187N/T189Q/L264R, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/V187T,A018K/G023Q/V077I/G156W, A018K/K024A/D130A/G156W,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/G163P/L227M/L264R,D027E/L227M/N233Q/L264R, D027E/S058M/G163P/L264R,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/N033D/E045F/N073R, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,D111A/D130A/V154I/G156W, D130A/G163P/L227M/L264R,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/A049V/V077I/G156W,G023Q/K024A/D130A/G156W, G023Q/K024A/L075R/G156W,G023Q/K024A/V077I/G156W, G023Q/L075Q/D130A/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/G091Q/V187N/L264R,L075R/D130A/V187T/L264R, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/N011K/D027S/P256T, S058M/D137Q/G163P/N233Q,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/L252Q/L264R,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D130A/G156W/V187N/L264R, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/K024A/N094R/G156W/V187T,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D027Q/F051T/L075Q/D130A/L264R,D027S/E056K/D111A/V187N/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/D137Q/N233Q/L264R, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E045F/A049V/L075Q/V187T/T189D, E056K/L075G/V187N/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,N011K/D027N/S058M/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, Q004D/N011K/D027N/N233Q/P256T,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,S058M/D137Q/G163P/L227M/L264R, S058M/D137Q/G163P/L227M/N233Q,S058M/G163P/L227M/N233Q/L264R, V077I/D130A/V154I/G156W/V187N,A018K/D027S/N033D/E045F/N073R/L075D,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 4-3 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value in the Thermostabilityassay compared to TLL SEQ ID NO: 1 are shown below. E045F/N073R,A018K/E045F/V187T, A018K/V154I/G156W, D111A/D130A/L264R,V187N/T189Q/L264R, A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/G023Q/V077I/G156W,A018K/D027S/E045F/L075D/T189D, A018K/D130A/G156W/V187N/L264R,A018K/G023K/D130A/V154I/G156W, A018K/G023Q/K024A/L075Q/G156W,D027S/E056K/D111A/V187N/L264R, G023K/K024A/D111A/D130A/V187T,G023K/L075Q/V187H/I252Q/L264R, N011K/G023K/D111A/G156W/L264R,S058M/D137Q/G163P/L227M/N233Q, S058M/G163P/L227M/N233Q/L264R,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,D048Q/S058M/D130A/D137Q/G163P/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/K024A/N094R/D130A/G156W/V187T/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 4-4 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 in the Thermostability assay are shown below.A018K/D027S, A018K/L075D, A018K/L075Q, A018K/T189D, D027E/G163P,D027S/N033D, D027S/P256T, D111A/L264R, D130A/L264R, D130A/T189Q,D130A/V187N, D130A/V187T, E045F/N073R, F051T/L075R, G023K/D130A,G023K/L264R, G023Q/L075Q, K024A/L075Q, K024A/V154I, L075Q/D130A,L075Q/G156W, L075R/L264R, L227M/L264R, N011K/D027S, N073R/L075D,P029E/N033D, S058M/L264R, V187T/L264R, A018K/A049V/L075Q,A018K/A049V/V187T, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/V187T, A018K/G023K/G156W, A018K/G023K/L075Q,A018K/G023Q/L075Q, A018K/K024A/V154I, A018K/L075D/T189D,A018K/L075Q/G156W, A018K/L075Q/V187T, A018K/N033D/L075D,A018K/N033D/T189D, A018K/N073R/L075D, A018K/P029E/T189D,A018K/V154I/G156W, D027E/S058M/G163P, D027N/N233Q/P256T,D027N/S058M/P256T, D027Q/N233Q/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/N073R/L075D,D027S/N233Q/P256T, D027S/S058M/P256T, D111A/D130A/L264R,D130A/T189Q/L264R, D130A/V154I/G156W, D130A/V187N/L264R,D130A/V187T/L264R, E045F/L075D/T189D, E045F/N073R/L075D,E056K/D130A/L264R, E056K/D130A/T189Q, G023K/D130A/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,G023Q/D111A/L264R, G023Q/D130A/G156W, G023Q/E056K/L075R,G023Q/K024A/G156W, G023Q/L075Q/V187T, G163P/L227M/L264R,K024A/D130A/V154I, K024A/L075Q/G156W, K024A/L075Q/V077I,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075Q/G156W/T189D, L075Q/G156W/V187N, L075Q/G156W/V187T,L075Q/N094R/V154I, L075Q/V077I/T189D, L075Q/V154I/V187T,L075Q/V187T/L264R, L075Q/V187T/T189D, L075R/D130A/L264R,L075R/D130A/V187T, L075R/V187N/L264R, L075R/V187T/L264R,N011K/D027S/S058M, N011K/N233Q/P256T, N073R/L075D/T189D,N094R/D130A/V187T, P029E/E045F/L075D, P029E/L075D/T189D,P029E/N033D/L075D, P029E/N073R/L075D, Q004D/D027S/P256T,Q004D/N233Q/P256T, S058M/L227M/L264R, V077I/D130A/V154I,V187N/T189Q/L264R, A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/N073R, A018K/D027S/E045F/T189D,A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/E045F/A049V/V187T,A018K/E045F/L075D/T189D, A018K/E045F/L075Q/V187T,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/T189D,A018K/G023Q/A049V/V187T, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/G156W/V187T,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V077I,A018K/G023Q/L075Q/V187T, A018K/G023Q/L075R/D130A,A018K/G023Q/V077I/G156W, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/K024A/D130A/G156W,A018K/L075Q/G156W/T189D, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D111A, A018K/L075Q/N094R/D130A,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/N033D/L075D/T189D, A018K/N073R/L075D/T189D,A018K/P029E/N033D/T189D, A018K/P029E/N073R/L075D,A049V/L075Q/V187T/T189D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, 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K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,A018K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187T/L264R,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/D027S/L075R/V154I/G156W/V187N/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023K/L075Q/D111A/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/D130A/V187T/T189Q/L264K,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 4-5 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 in theThermostability assay compared to Reference sequence SEQ ID NO: 2 areshown below. A018K/L075D, D027E/G163P, D027S/N033D, D027S/P256T,D111A/L264R, D130A/L264R, D130A/V187N, D130A/V187T, E045F/N073R,F051T/L075R, G023K/L264R, K024A/V154I, L075R/L264R, L227M/L264R,P029E/N033D, S058M/L264R, V187T/L264R, A018K/A049V/V187T,A018K/E045F/V187T, A018K/K024A/V154I, A018K/L075D/T189D,A018K/N033D/T189D, A018K/P029E/T189D, A018K/V154I/G156W,D027E/S058M/G163P, D027N/S058M/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/S058M/P256T,D111A/D130A/L264R, D130A/V154I/G156W, D130A/V187N/L264R,D130A/V187T/L264R, E056K/D130A/L264R, G023K/D130A/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,G023Q/D130A/G156W, G023Q/K024A/G156W, G163P/L227M/L264R,K024A/D130A/V154I, L075G/D130A/V187H, L075Q/V187T/L264R,L075R/D130A/L264R, L075R/D130A/V187T, L075R/V187N/L264R,L075R/V187T/L264R, N011K/N233Q/P256T, N094R/D130A/V187T,Q004D/D027S/P256T, V187N/T189Q/L264R, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/V187T,A018K/G023Q/V077I/G156W, A018K/K024A/D130A/G156W,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,A018K/P029E/N073R/L075D, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/G163P/L227M/L264R,D027E/L227M/N233Q/L264R, D027E/S058M/G163P/L264R,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/D130A/I252Q/L264R, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D048Q/G163P/N233Q/L264R, D111A/D130A/V154I/G156W,D130A/G163P/L227M/L264R, E045F/A049V/G156W/V187T,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/A049V/V077I/G156W,G023Q/K024A/L075R/G156W, G023Q/K024A/V077I/G156W,G023Q/L075Q/D130A/L264R, G091Q/V187T/I252Q/L264R,K024A/D130A/V154I/V187T, L075G/D130A/V187T/I252Q,L075G/G091Q/V187N/L264R, L075G/V187H/I252Q/L264R,L075R/D130A/V187T/L264R, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/N011K/D027S/P256T, S058M/D137Q/G163P/N233Q,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/I252Q/L264R,A018K/D027S/E045F/L075D/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D130A/G156W/V187N/L264R, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/K024A/N094R/G156W/V187T,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027E/S058M/G163P/L227M/L264R, D027Q/F051T/L075Q/D130A/L264R,D027S/E056K/D111A/V187N/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/D137Q/G163P/L227M/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/D137Q/N233Q/L264R, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E045F/A049V/L075Q/V187T/T189D, E056K/L075G/V187N/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,N011K/D027N/S058M/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,Q004D/N011K/D027N/N233Q/P256T, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/D137Q/G163P/L227M/L264R,S058M/D137Q/G163P/L227M/N233Q, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/G091Q/D130A/V187H/I252Q/L264R,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 4-6 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value in the Thermostability assay compared to Reference sequence SEQID NO: 2 are shown below. A018K/L075D, D027E/G163P, E045F/N073R,L227M/L264R, A018K/E045F/V187T, A018K/L075D/T189D, G163P/L227M/L264R,L075R/V187N/L264R, A018K/E045F/A049V/G156W, D027E/D048Q/G163P/L264R,D027E/D130A/N233Q/L264R, D027E/G163P/L227M/L264R,D027E/L227M/N233Q/L264R, D027E/S058M/G163P/L264R,D027S/P029E/N033D/E045F, D048Q/G163P/N233Q/L264R,D130A/G163P/L227M/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, G023K/D130A/V187T/L264R,G023Q/L075Q/D130A/L264R, L075G/G091Q/V187N/L264R,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,A018K/D027S/E045F/L075D/T189D, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D027E/S058M/D130A/G163P/L264R,D027E/S058M/G163P/L227M/L264R, D027S/E056K/D111A/V187N/L264R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D048Q/S058M/G163P/L227M/L264R, D048Q/S058M/G163P/N233Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/E056K/L075R/V187N/L264R,G023K/L075Q/V187H/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/L075Q/D130A/I252Q/L264R, L075G/D130A/V187T/I252Q/L264R,S058M/D130A/G163P/L227M/L264R, S058M/D137Q/G163P/L227M/L264R,S058M/D137Q/G163P/L227M/N233Q, D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/L075G/D130A/V187T/I252Q/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

Example 5 Detergent Stability of TLL Combinatorial Variants

The detergent stability of TLL variants created as described in Example2 was assayed as described in Example 1 (Detergent Stability assay). Theperformance index was calculated for the variants compared to TLL, SEQID NO:1 or the reference sequence SEQ ID NO: 2.

TABLE 5-1 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 in the Detergent stability assay are shown below. A018K/D027S,A018K/L075Q, A018K/T189D, D027E/G163P, D027N/E056K, D027N/N233Q,D027S/N033D, D027S/P256T, D111A/L264R, D130A/T189Q, D130A/V187N,G023K/D130A, G023Q/L075Q, K024A/L075Q, K024A/V154I, L075Q/D130A,L075Q/G156W, N011K/D027S, N073R/L075D, N094R/G156W, P029E/N033D,A018K/A049V/L075Q, A018K/A049V/V187T, A018K/D027S/E045F,A018K/D027S/N073R, A018K/D027S/T189D, A018K/E045F/T189D,A018K/E045F/V187T, A018K/G023K/G156W, A018K/G023K/L075Q,A018K/G023Q/L075Q, A018K/K024A/V154I, A018K/L075Q/G156W,A018K/L075Q/V187T, A018K/N033D/T189D, A018K/N073R/L075D,A018K/V154I/G156W, A049V/L075Q/T189D, A049V/V187T/T189D,D027E/S058M/G163P, D027N/N233Q/P256T, D027N/S058M/P256T,D027Q/N233Q/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D027S/N073R/L075D, D027S/N233Q/P256T,D027S/S058M/P256T, D111A/D130A/L264R, D130A/T189Q/L264R,D130A/V154I/G156W, D130A/V187T/L264R, E045F/L075D/T189D,E045F/N073R/L075D, E056K/D130A/L264R, G023K/D130A/L264R,G023K/D130A/V187T, G023K/V187N/L264R, G023K/V187T/L264R,G023Q/A049V/T189D, G023Q/D111A/L264R, G023Q/D130A/G156W,G023Q/E056K/L075R, G023Q/K024A/G156W, G023Q/L075Q/V187T,I090F/N233Q/P256T, K024A/D130A/V154I, K024A/L075Q/G156W,K024A/L075Q/V077I, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/D130A/V187T, L075Q/G156W/T189D, L075Q/G156W/V187N,L075Q/G156W/V187T, L075Q/V077I/T189D, L075Q/V154I/V187T,L075Q/V187N/L264R, L075Q/V187T/T189D, L075R/D130A/V187T,N011K/D027S/S058M, N011K/N233Q/P256T, N033D/E045F/L075D,N033D/N073R/T189D, N073R/L075D/T189D, N094R/D130A/V187T,P029E/E045F/L075D, P029E/L075D/T189D, P029E/N033D/E045F,P029E/N033D/L075D, P029E/N073R/L075D, Q004D/D027S/P256T,Q004D/S058M/N233Q, V077I/D130A/V154I, V077I/V187A/T189D,V187N/T189Q/L264R, A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/L075D/T189D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/E045F/A049V/G156W,A018K/E045F/A049V/V187T, A018K/E045F/L075Q/T189D,A018K/E045F/L075Q/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/T189D, A018K/G023Q/A049V/V187T,A018K/G023Q/E045F/T189D, A018K/G023Q/E045F/V187T,A018K/G023Q/G156W/V187T, A018K/G023Q/L075Q/T189D,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/G156W,A018K/G023Q/V077I/V187T, A018K/G023Q/V187T/T189D,A018K/K024A/D130A/G156W, A018K/L075Q/G156W/T189D,A018K/L075Q/G156W/V187T, A018K/L075Q/N094R/D111A,A018K/L075Q/N094R/D130A, A018K/L075Q/N094R/V187Q,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/L075D,A018K/P029E/N033D/T189D, A018K/V154I/G156W/V187T,A049V/L075Q/V077I/T189D, A049V/L075Q/V187T/T189D,D027E/D137Q/G163P/L227M, D027N/E056K/N233Q/P256T,D027N/E056K/S058M/P256T, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027Q/S058M/I090F/P256T,D027S/D130A/I252Q/L264R, D027S/E056K/D111A/V154I,D027S/E056K/I090F/P256T, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/L075D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/L075D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D027S/P029E/N033D/N073R, D027S/P029E/N033D/T189D,D111A/D130A/V154I/G156W, D111A/D130A/V154I/L264R,D111A/D130A/V187T/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, E056K/D130A/V187T/L264R,E056K/L075Q/V187N/L264R, G023K/D027Q/F051T/L075Q,G023K/D027S/L075Q/L264R, G023K/D111A/D130A/L264R,G023K/D130A/G156W/T189Q, G023K/D130A/V187N/L264R,G023K/D130A/V187T/L264R, G023K/D130A/V187T/T189Q,G023K/K024A/V154I/V187N, G023K/L075Q/D130A/V187N,G023K/L075Q/G156W/L264R, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023K/L075R/V187T/L264R,G023Q/A049V/L075Q/T189D, G023Q/A049V/V077I/G156W,G023Q/E045F/A049V/T189D, G023Q/E045F/A049V/V187T,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/K024A/D130A/G156W, G023Q/K024A/L075R/G156W,G023Q/K024A/L075R/V154I, G023Q/K024A/V077I/G156W,G023Q/L075Q/D130A/L264R, G023Q/L075Q/G156W/V187N,G023Q/L075Q/G156W/V187T, G023Q/L075Q/V187T/L264R,G023Q/V154I/G156W/V187N, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, K024A/L075Q/D130A/V154I,K024A/L075Q/V187T/T189Q, K024A/L075R/G156W/V187N,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/G156W/V187T/L264R,L075Q/V077I/D130A/V187Q, L075Q/V077I/G156W/V187N,L075Q/V077I/N094R/G156W, L075Q/V077I/V154I/V187Q,L075Q/V077I/V187T/T189D, L075R/D111A/V154I/G156W,L075R/D130A/V154I/L264R, N011K/D027N/E056K/S058M,N011K/D027N/S058M/P256T, N011K/D027Q/S058M/P256T,N011K/D027S/E056K/P256T, N011K/D027S/N233Q/P256T,N011K/D027S/S058M/N233Q, N011K/E056K/N233Q/P256T,N011K/G023K/L075Q/D111A, N011K/S058M/N233Q/P256T,P029E/E045F/L075D/T189D, P029E/E045F/N073R/L075D,P029E/E045F/N073R/T189D, P029E/N033D/E045F/L075D,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/D027S/I090F/P256T, Q004D/N011K/D027N/P256T,Q004D/N011K/D027Q/N233Q, Q004D/N011K/D027S/P256T,Q004D/N011K/E056K/S058M, S058M/D137Q/G163P/N233Q,S058M/L075Q/G091Q/I252Q, A018K/A049V/L075Q/V187T/T189D,A018K/D027S/N033D/L075D/T189D, A018K/D027S/N033D/N073R/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N033D/L075D,A018K/D027S/P029E/N073R/L075D, A018K/E045F/A049V/L075Q/T189D,A018K/E045F/A049V/L075Q/V187T, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/K024A/L075R/N094R, A018K/G023K/L075Q/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V077I, A018K/G023Q/A049V/L075Q/V187T,A018K/G023Q/A049V/V077I/V187T, A018K/G023Q/E045F/A049V/L075Q,A018K/G023Q/E045F/A049V/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/E045F/V077I/T189D, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/G023Q/L075Q/V187T/T189D,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/V154I/G156W,A018K/K024A/L075Q/V154I/T189D, A018K/K024A/L075R/D130A/V154I,A018K/K024A/L075R/N094R/D130A, A018K/K024A/N094R/G156W/V187T,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/G156W/V187T/T189D,A018K/L075Q/N094R/D130A/V187N, A018K/L075Q/N094R/G156W/V187N,A018K/L075Q/V077I/N094R/G156W, A018K/V077I/G156W/V187T/T189D,D027E/D048Q/L227M/N233Q/L264R, D027E/D048Q/S058M/G163P/N233Q,D027E/S058M/D137Q/G163P/N233Q, D027N/E056K/S058M/N233Q/P256T,D027Q/E056K/I090F/N233Q/P256T, D027Q/E056K/S058M/N233Q/P256T,D027S/E056K/D111A/V187N/L264R, D027S/E056K/S058M/N233Q/P256T,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075Q/D111A/D130A/V187N,D027S/N033D/N073R/L075D/T189D, D027S/P029E/E045F/N073R/T189D,D027S/P029E/N033D/E045F/N073R, D027S/P029E/N033D/N073R/L075D,D027S/P029E/N073R/L075D/T189D, D048Q/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E045F/A049V/L075Q/V187T/T189D, E056K/D130A/V187H/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, E056K/L075R/D130A/V187T/L264R,E056K/N094R/G156W/V187N/L264R, E056K/S058M/I090F/N233Q/P256T,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/D111A/V154I/V187T/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/D130A/V154I/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/A049V/L075Q/G156W/T189D, G023Q/A049V/L075Q/G156W/V187T,G023Q/A049V/L075Q/V187T/T189D, G023Q/D027S/V154I/V187N/L264R,G023Q/E045F/A049V/G156W/T189D, G023Q/E045F/A049V/L075Q/V077I,G023Q/E045F/A049V/V077I/T189D, G023Q/E045F/L075Q/G156W/T189D,G023Q/E045F/L075Q/V077I/V187T, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/D130A/G156W, G023Q/K024A/L075Q/G156W/V187N,G023Q/K024A/L075Q/G156W/V187Q, G023Q/K024A/L075Q/V077I/V187Q,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/G156W/V187Q,G023Q/K024A/L075R/V077I/D130A, G023Q/K024A/V077I/D130A/V154I,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,G023Q/L075Q/V077I/G156W/V187N, K024A/L075Q/D111A/G156W/V187T,K024A/L075Q/V077I/G156W/V187N, K024A/L075Q/V077I/V154I/V187N,K024A/L075R/D111A/V154I/V187T, K024A/L075R/G156W/V187N/T189Q,K024A/L075R/V154I/G156W/V187Q, L075Q/D111A/V187N/T189Q/L264R,L075Q/D130A/V187T/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075Q/V154I/V187N/T189Q/L264R,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,L075R/G156W/V187T/T189Q/L264R, L075R/V077I/N094R/V154I/G156W,N011K/D027N/E056K/N233Q/P256T, N011K/D027N/E056K/S058M/P256T,N011K/D027N/S058M/N233Q/P256T, N011K/D027Q/E056K/S058M/N233Q,N011K/D027Q/I090F/N233Q/P256T, N011K/D027Q/S058M/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, N011K/G023K/E056K/L075Q/T189Q,N011K/G023Q/L075Q/V187N/T189Q, P029E/N033D/E045F/L075D/T189D,P029E/N033D/K074S/L075D/T189D, P029E/N033D/N073R/L075D/T189D,Q004D/D027N/E056K/S058M/P256T, Q004D/D027N/I090F/N233Q/P256T,Q004D/D027Q/E056K/N233Q/P256T, Q004D/D027Q/E056K/S058M/P256T,Q004D/D027S/E056K/N233Q/P256T, Q004D/D027S/E056K/S058M/P256T,Q004D/D027S/S058M/N233Q/P256T, Q004D/N011K/D027N/N233Q/P256T,Q004D/N011K/D027Q/E056K/N233Q, Q004D/N011K/D027Q/S058M/N233Q,Q004D/N011K/I090F/N233Q/P256T, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, 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A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/D027S/L075R/V154I/G156W/V187N/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 5-2 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 in the Detergent stability assay compared to TLLSEQ ID NO: 1 are shown below. D027E/G163P, D027S/N033D, D027S/P256T,D111A/L264R, D130A/V187N, K024A/V154I, N094R/G156W, P029E/N033D,A018K/A049V/V187T, A018K/E045F/V187T, A018K/K024A/V154I,A018K/N033D/T189D, A018K/V154I/G156W, D027E/S058M/G163P,D027N/S058M/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D027S/S058M/P256T, D111A/D130A/L264R,D130A/V154I/G156W, D130A/V187T/L264R, E056K/D130A/L264R,G023K/D130A/L264R, G023K/D130A/V187T, G023K/V187N/L264R,G023K/V187T/L264R, G023Q/A049V/T189D, G023Q/D130A/G156W,G023Q/K024A/G156W, I090F/N233Q/P256T, K024A/D130A/V154I,L075G/D130A/V187H, L075Q/V187N/L264R, L075R/D130A/V187T,N011K/N233Q/P256T, N094R/D130A/V187T, P029E/N033D/E045F,Q004D/D027S/P256T, Q004D/S058M/N233Q, V187N/T189Q/L264R,A018K/D027S/P029E/T189D, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/E045F/A049V/G156W,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/V187T,A018K/G023Q/V077I/G156W, A018K/K024A/D130A/G156W,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,D027E/D137Q/G163P/L227M, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D111A/D130A/V154I/G156W,D111A/D130A/V187T/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, E056K/D130A/V187T/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/K024A/V154I/V187N,G023K/L075R/D130A/L264R, G023K/L075R/V187T/L264R,G023Q/A049V/V077I/G156W, G023Q/F051T/I252Q/L264R,G023Q/G091Q/I252Q/L264R, G023Q/K024A/D130A/G156W,G023Q/K024A/L075R/G156W, G023Q/K024A/V077I/G156W,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,K024A/D130A/V154I/V187T, L075G/D130A/V187T/I252Q,N011K/D027Q/S058M/P256T, N011K/D027S/N233Q/P256T,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/N011K/D027S/P256T,S058M/D137Q/G163P/N233Q, S058M/L075Q/G091Q/I252Q,A018K/D027S/P029E/N033D/L075D, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/G156W/V187T, D027E/S058M/D137Q/G163P/N233Q,D027S/E056K/D111A/V187N/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E045F/A049V/L075Q/V187T/T189D, E056K/L075R/D130A/V187T/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/D111A/G156W/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187H/I252Q/L264R,G023K/L075R/D130A/V187N/L264R, G023K/L075R/D130A/V187T/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/V154I/G156W/V187Q,N011K/D027N/S058M/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, Q004D/N011K/D027N/N233Q/P256T,S058M/G163P/L227M/N233Q/L264R, V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 5-3 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value in the Detergentstability assay compared to TLL SEQ ID NO: 1 are shown below.D027S/P256T, K024A/V154I, A018K/A049V/V187T, A018K/K024A/V154I,A018K/V154I/G156W, D027N/S058M/P256T, D027S/S058M/P256T,G023Q/A049V/T189D, L075Q/V187N/L264R, N011K/N233Q/P256T,P029E/N033D/E045F, Q004D/D027S/P256T, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/V077I/G156W,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/N033D/E045F/T189D, D111A/D130A/V154I/G156W,E045F/L075Q/G156W/V187T, G023K/D130A/G156W/T189Q,G023K/D130A/V187T/T189Q, G023K/K024A/V154I/V187N,G023Q/A049V/V077I/G156W, G023Q/K024A/L075R/G156W,K024A/D130A/V154I/V187T, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, Q004D/D027N/E056K/P256T,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/N011K/D027S/P256T,A018K/G023K/D130A/V154I/G156W, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/L075Q/G156W, D027S/E056K/D111A/V187N/L264R,D130A/V154I/G156W/V187N/T189Q, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/D027N/S058M/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,Q004D/N011K/D027N/N233Q/P256T, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,D048Q/S058M/D130A/D137Q/G163P/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 5-4 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 in the Detergent stability assay are shown below.A018K/D027S, A018K/L075D, A018K/L075Q, D027E/G163P, D027N/E056K,D027N/N233Q, D027S/N033D, D027S/P256T, D111A/L264R, D130A/L264R,G023K/D130A, G023Q/L075Q, K024A/L075Q, K024A/V154I, L075Q/D130A,L075Q/G156W, N073R/L075D, P029E/N033D, A018K/A049V/L075Q,A018K/A049V/V187T, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/T189D, A018K/G023K/G156W, A018K/G023K/L075Q,A018K/G023Q/L075Q, A018K/K024A/V154I, A018K/L075D/T189D,A018K/L075Q/G156W, A018K/L075Q/V187T, A018K/N033D/L075D,A018K/N033D/T189D, A018K/N073R/L075D, A018K/P029E/T189D,A018K/V154I/G156W, A049V/L075Q/T189D, A049V/V187T/T189D,D027E/S058M/G163P, D027N/N233Q/P256T, D027N/S058M/P256T,D027Q/N233Q/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D027S/N073R/L075D, D027S/N233Q/P256T,D027S/S058M/P256T, D130A/V187T/L264R, E045F/L075D/T189D,E045F/N073R/L075D, E056K/D130A/L264R, G023K/D130A/L264R,G023K/D130A/V187T, G023K/V187N/L264R, G023K/V187T/L264R,G023Q/A049V/T189D, G023Q/E056K/L075R, G023Q/L075Q/V187T,I090F/N233Q/P256T, K024A/L075Q/G156W, K024A/L075Q/V077I,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075Q/G156W/T189D, L075Q/G156W/V187N, L075Q/G156W/V187T,L075Q/V077I/T189D, L075Q/V154I/V187T, L075Q/V187N/L264R,L075Q/V187T/T189D, L075R/D130A/V187T, N011K/D027S/S058M,N011K/N233Q/P256T, N033D/E045F/L075D, N033D/N073R/T189D,N073R/L075D/T189D, N094R/D130A/V187T, P029E/E045F/L075D,P029E/L075D/T189D, P029E/N033D/E045F, P029E/N033D/L075D,P029E/N073R/L075D, Q004D/D027S/P256T, S058M/L227M/L264R,V077I/D130A/V154I, A018K/A049V/L075Q/T189D, A018K/D027S/L075D/T189D,A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/T189D, A018K/G023Q/A049V/V187T,A018K/G023Q/E045F/T189D, A018K/G023Q/E045F/V187T,A018K/G023Q/G156W/V187T, A018K/G023Q/L075Q/T189D,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/G156W,A018K/G023Q/V077I/V187T, A018K/G023Q/V187T/T189D,A018K/L075Q/G156W/T189D, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D130A, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/L075D,A018K/P029E/N033D/T189D, A018K/V154I/G156W/V187T,A049V/L075Q/V077I/T189D, A049V/L075Q/V187T/T189D,D027E/D137Q/G163P/L227M, D027N/E056K/N233Q/P256T,D027N/E056K/S058M/P256T, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027S/D130A/I252Q/L264R,D027S/E056K/D111A/V154I, D027S/E056K/I090F/P256T,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/L075D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/L075D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D027S/P029E/N033D/N073R,D027S/P029E/N033D/T189D, D111A/D130A/V154I/G156W,D111A/D130A/V154I/L264R, E045F/L075Q/G156W/V187T,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,E056K/L075Q/V187N/L264R, F051T/L075Q/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D027S/L075Q/L264R,G023K/D111A/D130A/L264R, G023K/D130A/G156W/T189Q,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/K024A/V154I/V187N,G023K/L075Q/D130A/V187N, G023K/L075Q/G156W/L264R,G023K/L075Q/G156W/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/A049V/L075Q/T189D,G023Q/A049V/V077I/G156W, G023Q/E045F/A049V/T189D,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/K024A/L075R/G156W, G023Q/K024A/L075R/V154I,G023Q/K024A/V077I/G156W, G023Q/L075Q/D130A/L264R,G023Q/L075Q/G156W/V187N, G023Q/L075Q/G156W/V187T,G023Q/L075Q/V187T/L264R, G023Q/V154I/G156W/V187N,K024A/D130A/V154I/V187T, K024A/L075Q/D130A/G156W,K024A/L075Q/D130A/V154I, K024A/L075Q/V187T/T189Q,K024A/L075R/G156W/V187N, L075G/D130A/V187T/I252Q,L075G/G091Q/V187N/L264R, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/G156W/V187T/L264R,L075Q/V077I/D130A/V187Q, L075Q/V077I/G156W/V187N,L075Q/V077I/N094R/G156W, L075Q/V077I/V154I/V187Q,L075Q/V077I/V187T/T189D, L075R/D111A/V154I/G156W,L075R/D130A/V154I/L264R, N011K/D027N/E056K/S058M,N011K/D027Q/S058M/P256T, N011K/D027S/E056K/P256T,N011K/D027S/N233Q/P256T, N011K/D027S/S058M/N233Q,N011K/E056K/N233Q/P256T, N011K/G023K/L075Q/D111A,N011K/S058M/N233Q/P256T, P029E/E045F/L075D/T189D,P029E/E045F/N073R/L075D, P029E/N033D/E045F/L075D,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/D027S/I090F/P256T, Q004D/N011K/D027N/P256T,Q004D/N011K/D027S/P256T, S058M/D137Q/G163P/N233Q,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/I252Q/L264R,A018K/A049V/L075Q/V187T/T189D, A018K/D027S/N033D/L075D/T189D,A018K/D027S/N033D/N073R/T189D, A018K/D027S/P029E/E045F/T189D,A018K/D027S/P029E/N033D/L075D, A018K/D027S/P029E/N073R/L075D,A018K/E045F/A049V/L075Q/T189D, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/K024A/L075R/N094R, A018K/G023K/L075Q/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/G023Q/L075Q/V187T/T189D,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/V154I/G156W,A018K/K024A/L075Q/V154I/T189D, A018K/K024A/L075R/D130A/V154I,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/G156W/V187T/T189D,A018K/L075Q/V077I/N094R/G156W, A018K/V077I/G156W/V187T/T189D,D027E/D048Q/G163P/N233Q/L264R, D027E/D048Q/L227M/N233Q/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D137Q/G163P/N233Q,D027N/E056K/S058M/N233Q/P256T, D027Q/E056K/I090F/N233Q/P256T,D027Q/E056K/S058M/N233Q/P256T, D027S/E056K/D111A/V187N/L264R,D027S/E056K/S058M/N233Q/P256T, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/N033D/N073R/L075D/T189D, D027S/P029E/N033D/N073R/L075D,D027S/P029E/N073R/L075D/T189D, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/N233E,D048Q/S058M/D137Q/N233Q/L264R, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/D130A/V187H/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, E056K/L075R/D130A/V187T/L264R,E056K/N094R/G156W/V187N/L264R, E056K/S058M/I090F/N233Q/P256T,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075Q/G091Q/D130A/L264R, F051T/L075Q/V187N/I252Q/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/D111A/V154I/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/D111A/G156W/T189Q,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/D130A/G156W/L264R, G023K/L075Q/D130A/V154I/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187H/I252Q/L264R,G023K/L075R/D130A/V187N/L264R, G023K/L075R/D130A/V187T/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/T189D,G023Q/A049V/L075Q/G156W/V187T, G023Q/A049V/L075Q/V187T/T189D,G023Q/D027S/L075G/I252Q/L264R, G023Q/D027S/V154I/V187N/L264R,G023Q/E045F/A049V/G156W/T189D, G023Q/E045F/L075Q/G156W/T189D,G023Q/E045F/L075Q/V077I/V187T, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187N, G023Q/K024A/L075Q/G156W/V187Q,G023Q/K024A/L075Q/V077I/V187Q, G023Q/K024A/L075R/D130A/V154I,G023Q/K024A/L075R/G156W/V187Q, G023Q/K024A/L075R/V077I/D130A,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,G023Q/L075Q/V077I/G156W/V187N, G091Q/D130A/V187H/I252Q/L264R,K024A/L075Q/D111A/G156W/V187T, K024A/L075Q/V077I/G156W/V187N,K024A/L075Q/V077I/V154I/V187N, K024A/L075R/D111A/V154I/V187T,K024A/L075R/G156W/V187N/T189Q, K024A/L075R/V154I/G156W/V187Q,L075Q/D111A/V187N/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075Q/V154I/V187N/T189Q/L264R,L075R/D130A/V154I/T189Q/L264R, L075R/G156W/V187T/T189Q/L264R,L075R/V077I/N094R/V154I/G156W, N011K/D027N/E056K/N233Q/P256T,N011K/D027N/E056K/S058M/P256T, N011K/D027N/S058M/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q, N011K/D027Q/I090F/N233Q/P256T,N011K/D027Q/S058M/N233Q/P256T, N011K/D027S/E056K/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,N011K/G023K/E056K/L075Q/T189Q, N011K/G023Q/L075Q/V187N/T189Q,P029E/N033D/K074S/L075D/T189D, P029E/N033D/N073R/L075D/T189D,Q004D/D027N/E056K/S058M/P256T, Q004D/D027Q/E056K/N233Q/P256T,Q004D/D027Q/E056K/S058M/P256T, Q004D/D027S/E056K/N233Q/P256T,Q004D/D027S/E056K/S058M/P256T, Q004D/D027S/S058M/N233Q/P256T,Q004D/N011K/D027N/N233Q/P256T, Q004D/N011K/D027Q/E056K/N233Q,Q004D/N011K/I090F/N233Q/P256T, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, 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S/L075R/V154I/G156W/V187N/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 5-5 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 in the Detergentstability assay compared to Reference sequence SEQ ID NO: 2 are shownbelow. A018K/L075D, D027E/G163P, D027S/N033D, D027S/P256T, D111A/L264R,D130A/L264R, K024A/V154I, P029E/N033D, A018K/A049V/V187T,A018K/K024A/V154I, A018K/L075D/T189D, A018K/N033D/T189D,A018K/P029E/T189D, A018K/V154I/G156W, D027E/S058M/G163P,D027N/S058M/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D027S/S058M/P256T, D130A/V187T/L264R,E056K/D130A/L264R, G023K/D130A/L264R, G023K/D130A/V187T,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,I090F/N233Q/P256T, L075G/D130A/V187H, L075Q/V187N/L264R,L075R/D130A/V187T, N011K/N233Q/P256T, N094R/D130A/V187T,P029E/N033D/E045F, Q004D/D027S/P256T, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/L075D/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/V077I/G156W,A018K/N033D/L075D/T189D, D027E/D137Q/G163P/L227M,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/D130A/I252Q/L264R, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D111A/D130A/V154I/G156W, E045F/L075Q/G156W/V187T,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/L075Q/I252Q/L264R, G023K/D027Q/F051T/L075Q,G023K/D130A/G156W/T189Q, G023K/D130A/V187N/L264R,G023K/D130A/V187T/L264R, G023K/D130A/V187T/T189Q,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/A049V/V077I/G156W,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/K024A/L075R/G156W, G023Q/K024A/V077I/G156W,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,K024A/D130A/V154I/V187T, L075G/D130A/V187T/I252Q,L075G/G091Q/V187N/L264R, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, P029E/N073R/L075D/T189D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/N011K/D027S/P256T, S058M/D137Q/G163P/N233Q,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/I252Q/L264R,A018K/D027S/P029E/N033D/L075D, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/L075R/D130A/V154I, D027E/D048Q/G163P/N233Q/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D137Q/G163P/N233Q,D027S/E056K/D111A/V187N/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/D137Q/N233Q/L264R,D048Q/S058M/G163P/L227M/L264R, D048Q/S058M/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/L075R/D130A/V187T/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/D111A/G156W/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187H/I252Q/L264R,G023K/L075R/D130A/V187N/L264R, G023K/L075R/D130A/V187T/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/D027S/L075G/I252Q/L264R,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/D027N/S058M/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,Q004D/N011K/D027N/N233Q/P256T, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 5-6 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value in the Detergent stability assay compared to Reference sequenceSEQ ID NO: 2 are shown below. D027S/N033D, D027S/L075Q/G091Q,D027S/N033D/T189D, E056K/D130A/L264R, G023K/D130A/L264R,G023K/D130A/V187T, G023K/V187N/L264R, G023K/V187T/L264R,L075G/D130A/V187H, A018K/D027S/P029E/T189D, D027Q/E056K/N233Q/P256T,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, E056K/D130A/V187T/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/V187N/L264R,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, L075G/D130A/V187T/I252Q,P029E/N073R/L075D/T189D, Q004D/D027Q/N233Q/P256T,Q004D/N011K/D027S/P256T, A018K/D027S/P029E/N033D/L075D,A018K/G023K/D130A/V154I/G156W, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187/I252Q/L264R,G023K/L075R/D130A/V187N/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/L075G/G091Q/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,A018K/G023K/L075Q/V077I/G156W/V187Q,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/L075G/G091Q/D130A/I252Q/L264R,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q

Example 6 LAS Stability of TLL Combinatorial Variants

The LAS-stability of TLL variants created as described in Example 2 wasassayed as described in Example 1 (LAS-stability assay). The performanceindex was calculated for the variants compared to TLL, SEQ ID NO:1 orthe reference sequence SEQ ID NO: 2.

TABLE 6-1 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 in the LAS stability assay are shown below. A018K/L075Q,A018K/T189D, D027E/G163P, D027N/E056K, D027N/N233Q, D027S/N033D,D027S/P256T, D111A/L264R, D130A/T189Q, D130A/V187N, D130A/V187T,G023K/D130A, G023Q/L075Q, K024A/L075Q, K024A/V154I, L075Q/D130A,L075Q/G156W, N011K/D027S, N073R/L075D, N094R/G156W, P029E/N033D,A018K/A049V/L075Q, A018K/A049V/V187T, A018K/D027S/E045F,A018K/D027S/N073R, A018K/D027S/T189D, A018K/E045F/T189D,A018K/E045F/V187T, A018K/G023K/G156W, A018K/G023K/L075Q,A018K/G023Q/L075Q, A018K/K024A/V154I, A018K/L075Q/G156W,A018K/L075Q/V187T, A018K/N033D/T189D, A018K/N073R/L075D,A018K/P029E/T189D, A018K/V154I/G156W, A049V/V187T/T189D,D027E/S058M/G163P, D027N/N233Q/P256T, D027N/S058M/P256T,D027Q/N233Q/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D027S/N073R/L075D, D027S/N233Q/P256T,D027S/S058M/P256T, D111A/D130A/L264R, D130A/V154I/G156W,E045F/L075D/T189D, E056K/D130A/L264R, G023K/D130A/V187T,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,G023Q/D130A/G156W, G023Q/E056K/L075R, G023Q/K024A/G156W,G023Q/L075Q/V187T, I090F/N233Q/P256T, K024A/D130A/V154I,K024A/L075Q/G156W, K024A/L075Q/V077I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/D130A/V187T, L075Q/G156W/T189D,L075Q/G156W/V187N, L075Q/G156W/V187T, L075Q/N094R/V154I,L075Q/V077I/T189D, L075Q/V154I/V187T, L075Q/V187N/L264R,L075Q/V187T/T189D, L075R/D130A/L264R, L075R/D130A/V187T,N011K/D027S/S058M, N011K/N233Q/P256T, N033D/E045F/L075D,N033D/N073R/T189D, N073R/L075D/T189D, N094R/D130A/V187T,P029E/L075D/T189D, P029E/N033D/E045F, P029E/N033D/L075D,P029E/N073R/L075D, Q004D/D027S/P256T, Q004D/S058M/N233Q,V077I/D130A/V154I, V077I/V187A/T189D, A018K/A049V/L075Q/T189D,A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/E045F/A049V/V187T,A018K/E045F/L075Q/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/T189D, A018K/G023Q/A049V/V187T,A018K/G023Q/E045F/T189D, A018K/G023Q/E045F/V187T,A018K/G023Q/G156W/V187T, A018K/G023Q/L075Q/T189D,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/G156W,A018K/G023Q/V077I/V187T, A018K/G023Q/V187T/T189D,A018K/K024A/D130A/G156W, A018K/L075Q/G156W/T189D,A018K/L075Q/G156W/V187T, A018K/L075Q/N094R/D111A,A018K/L075Q/N094R/D130A, A018K/L075Q/N094R/V187Q,A018K/L075Q/V077I/N094R, A018K/L075Q/V187T/T189D,A018K/N033D/L075D/T189D, A018K/N073R/L075D/T189D,A018K/P029E/N033D/L075D, A018K/P029E/N033D/T189D,A049V/L075Q/V077I/T189D, A049V/L075Q/V187T/T189D,D027N/E056K/N233Q/P256T, D027N/E056K/S058M/P256T,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E056K/D111A/V154I, D027S/E056K/I090F/P256T,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/L075D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/L075D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D027S/P029E/N033D/N073R,D027S/P029E/N033D/T189D, D048Q/D137Q/G163P/L227M,D111A/D130A/V154I/G156W, D111A/D130A/V154I/L264R,D111A/D130A/V187T/L264R, E045F/L075Q/G156W/V187T,E056K/D130A/V187T/L264R, E056K/L075Q/V187N/L264R,F051T/D130A/I252Q/L264R, G023K/D027Q/F051T/L075Q,G023K/D027S/L075Q/L264R, G023K/D111A/D130A/L264R,G023K/D130A/G156W/T189Q, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023K/L075Q/D130A/V187N,G023K/L075Q/G156W/L264R, G023K/L075Q/G156W/V187N,G023Q/A049V/L075Q/T189D, G023Q/A049V/L075Q/V077I,G023Q/A049V/V077I/G156W, G023Q/D027S/D111A/G156W,G023Q/E045F/A049V/T189D, G023Q/E045F/A049V/V187T,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/K024A/D130A/G156W, G023Q/K024A/L075R/G156W,G023Q/K024A/L075R/V154I, G023Q/K024A/V077I/G156W,G023Q/L075Q/G156W/V187N, G023Q/L075Q/G156W/V187T,G023Q/V154I/G156W/V187N, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, K024A/L075Q/D130A/V154I,K024A/L075Q/V187T/T189Q, K024A/L075R/G156W/V187N,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/G156W/V187T/L264R,L075Q/V077I/D130A/V187Q, L075Q/V077I/G156W/V187N,L075Q/V077I/N094R/G156W, L075Q/V077I/V154I/V187Q,L075Q/V077I/V187T/T189D, L075R/D111A/V154I/G156W,L075R/D130A/V154I/L264R, N011K/D027N/E056K/S058M,N011K/D027N/S058M/P256T, N011K/D027Q/S058M/P256T,N011K/D027S/E056K/P256T, N011K/D027S/N233Q/P256T,N011K/E056K/N233Q/P256T, N011K/E056K/S058M/P256T,N011K/G023K/L075Q/D111A, N011K/S058M/N233Q/P256T,P029E/E045F/L075D/T189D, P029E/E045F/N073R/L075D,P029E/E045F/N073R/T189D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/D027S/I090F/P256T,Q004D/N011K/D027N/P256T, Q004D/N011K/D027Q/N233Q,Q004D/N011K/D027S/P256T, S058M/L075Q/G091Q/I252Q,A018K/A049V/L075Q/V187T/T189D, A018K/D027S/N033D/L075D/T189D,A018K/D027S/N033D/N073R/T189D, A018K/D027S/P029E/E045F/T189D,A018K/D027S/P029E/N033D/L075D, A018K/D027S/P029E/N073R/L075D,A018K/E045F/A049V/L075Q/G156W, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/K024A/L075R/N094R, A018K/G023K/L075Q/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V077I, A018K/G023Q/A049V/L075Q/V187T,A018K/G023Q/A049V/V077I/V187T, A018K/G023Q/E045F/A049V/L075Q,A018K/G023Q/E045F/A049V/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/K024A/L075Q/G156W, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/V187T/T189D,A018K/K024A/L075Q/V154I/G156W, A018K/K024A/L075Q/V154I/T189D,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/K024A/N094R/G156W/V187T, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/G156W/V187T/T189D, A018K/L075Q/N094R/D130A/V187N,A018K/L075Q/N094R/G156W/V187N, A018K/L075Q/V077I/N094R/G156W,A018K/V077I/G156W/V187T/T189D, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/D048Q/S058M/G163P/N233Q,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D137Q/G163P/N233Q,D027N/E056K/S058M/N233Q/P256T, D027N/S058M/I090F/N233Q/P256T,D027Q/E056K/I090F/N233Q/P256T, D027Q/E056K/S058M/N233Q/P256T,D027S/E056K/D111A/V187N/L264R, D027S/E056K/S058M/N233Q/P256T,D027S/L075R/V154I/T189Q/L264R, D027S/N033D/N073R/L075D/T189D,D027S/P029E/E045F/N073R/T189D, D027S/P029E/N033D/N073R/L075D,D027S/P029E/N073R/L075D/T189D, D048Q/S058M/G163P/N233Q/L264R,D130A/G163P/L227M/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/D130A/V187H/I252Q/L264R, E056K/L075Q/G156W/T189Q/L264R,E056K/L075R/D130A/V187T/L264R, E056K/N094R/G156W/V187N/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/D111A/V154I/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/D111A/G156W/T189Q, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/D130A/V154I/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/V187T,G023Q/A049V/L075Q/V187T/T189D, G023Q/D027S/D111A/T189Q/L264R,G023Q/D027S/L075G/I252Q/L264R, G023Q/D027S/V154I/V187N/L264R,G023Q/E045F/A049V/G156W/T189D, G023Q/E045F/L075Q/G156W/T189D,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/D130A/G156W,G023Q/K024A/L075Q/G156W/V187N, G023Q/K024A/L075Q/G156W/V187Q,G023Q/K024A/L075Q/V077I/V187Q, G023Q/K024A/L075R/D130A/V154I,G023Q/K024A/L075R/G156W/V187Q, G023Q/K024A/L075R/V077I/D130A,G023Q/K024A/V077I/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,G023Q/L075Q/V077I/G156W/V187N, K024A/L075Q/D111A/G156W/V187T,K024A/L075Q/V077I/G156W/V187N, K024A/L075Q/V077I/V154I/V187N,K024A/L075R/D111A/V154I/V187T, K024A/L075R/G156W/V187N/T189Q,K024A/L075R/V154I/G156W/V187Q, L075Q/D111A/D130A/V187T/T189Q,L075Q/D111A/V187N/T189Q/L264R, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075Q/V077I/D130A/G156W/V187N,L075Q/V154I/V187N/T189Q/L264R, L075R/D111A/D130A/V187N/T189Q,L075R/D130A/V154I/T189Q/L264R, L075R/V077I/N094R/V154I/G156W,N011K/D027N/E056K/N233Q/P256T, N011K/D027N/E056K/S058M/P256T,N011K/D027N/S058M/N233Q/P256T, N011K/D027Q/E056K/S058M/N233Q,N011K/D027Q/I090F/N233Q/P256T, N011K/D027Q/S058M/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, N011K/G023K/E056K/L075Q/T189Q,N011K/G023Q/L075Q/V187N/T189Q, P029E/N033D/K074S/L075D/T189D,P029E/N033D/N073R/L075D/T189D, Q004D/D027N/E056K/S058M/P256T,Q004D/D027N/I090F/N233Q/P256T, Q004D/D027Q/E056K/N233Q/P256T,Q004D/D027Q/E056K/S058M/P256T, Q004D/D027S/E056K/N233Q/P256T,Q004D/D027S/S058M/N233Q/P256T, Q004D/N011K/D027N/N233Q/P256T,Q004D/N011K/D027Q/E056K/N233Q, Q004D/N011K/D027Q/S058M/N233Q,S058M/G163P/L227M/N233Q/L264R, 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R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 6-2 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 in the LAS stability assay compared to TLL SEQ IDNO: 1 are shown below. D027E/G163P, D027S/N033D, D027S/P256T,D111A/L264R, D130A/V187N, D130A/V187T, K024A/V154I, N094R/G156W,P029E/N033D, A018K/A049V/V187T, A018K/E045F/V187T, A018K/K024A/V154I,A018K/N033D/T189D, A018K/P029E/T189D, A018K/V154I/G156W,D027E/S058M/G163P, D027N/S058M/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/S058M/P256T,D111A/D130A/L264R, D130A/V154I/G156W, E056K/D130A/L264R,G023K/D130A/V187T, G023K/V187N/L264R, G023K/V187T/L264R,G023Q/A049V/T189D, G023Q/D130A/G156W, G023Q/K024A/G156W,I090F/N233Q/P256T, K024A/D130A/V154I, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/L264R, L075R/D130A/V187T,N011K/N233Q/P256T, N094R/D130A/V187T, P029E/N033D/E045F,Q004D/D027S/P256T, Q004D/S058M/N233Q, A018K/D027S/P029E/T189D,A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/V077I/G156W,A018K/K024A/D130A/G156W, A018K/L075Q/V077I/N094R,A018K/N033D/L075D/T189D, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D137Q/G163P/L227M, D111A/D130A/V154I/G156W,D111A/D130A/V187T/L264R, E045F/L075Q/G156W/V187T,E056K/D130A/V187T/L264R, F051T/D130A/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/D130A/V187T/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/L075R/L264R, G023K/K024A/V154I/V187N,G023Q/A049V/V077I/G156W, G023Q/K024A/D130A/G156W,G023Q/K024A/L075R/G156W, G023Q/K024A/V077I/G156W,K024A/D130A/V154I/V187T, L075G/D130A/V187T/I252Q,N011K/D027Q/S058M/P256T, N011K/D027S/N233Q/P256T,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/N011K/D027S/P256T,S058M/L075Q/G091Q/I252Q, A018K/D027S/P029E/N033D/L075D,A018K/G023K/D130A/V154I/G156W, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/K024A/N094R/G156W/V187T, D027E/D048Q/D137Q/L227M/L264R,D027E/D048Q/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q, D027S/E056K/D111A/V187N/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/S058M/G163P/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/L075R/D130A/V187T/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/D111A/G156W/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187H/I252Q/L264R,G023K/L075R/D130A/V187N/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/D027S/L075G/I252Q/L264R, G023Q/F051T/L075Q/G091Q/I252Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/D027N/S058M/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, N011K/G023K/D111A/G156W/L264R,Q004D/N011K/D027N/N233Q/P256T, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 6-3 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value in the LAS stabilityassay compared to TLL SEQ ID NO: 1 are shown below. D027S/P256T,D130A/V187T, K024A/V154I, A018K/A049V/V187T, A018K/E045F/V187T,A018K/K024A/V154I, A018K/V154I/G156W, D027N/S058M/P256T,D027Q/S058M/P256T, D027S/L075Q/G091Q, D027S/N033D/T189D,D027S/S058M/P256T, D130A/V154I/G156W, G023K/D130A/V187T,G023K/V187T/L264R, G023Q/A049V/T189D, I090F/N233Q/P256T,K024A/D130A/V154I, L075Q/V187N/L264R, L075R/D130A/L264R,N011K/N233Q/P256T, N094R/D130A/V187T, Q004D/D027S/P256T,Q004D/S058M/N233Q, A018K/D027S/P029E/T189D, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/G023Q/V077I/G156W,A018K/K024A/D130A/G156W, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D137Q/G163P/L227M, D111A/D130A/V154I/G156W,E045F/L075Q/G156W/V187T, F051T/D130A/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023Q/A049V/V077I/G156W,G023Q/K024A/D130A/G156W, G023Q/K024A/L075R/G156W,G023Q/K024A/V077I/G156W, K024A/D130A/V154I/V187T,N011K/D027Q/S058M/P256T, N011K/D027S/N233Q/P256T,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/N011K/D027S/P256T, S058M/L075Q/G091Q/I252Q,A018K/D027S/P029E/N033D/L075D, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/K024A/L075Q/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/G156W/V187T, D027E/D048Q/D137Q/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027S/E056K/D111A/V187N/L264R,D027S/L075R/V154I/T189Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/L075R/D130A/V187T/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/D111A/G156W/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/D027S/L075G/I252Q/L264R,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/V154I/G156W/V187Q,N011K/D027N/S058M/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/D111A/G156W/L264R, Q004D/N011K/D027N/N233Q/P256T,V077I/D130A/V154I/G156W/V187N, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/N094R/D111A/D130A/V154I/V187N,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 6-4 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 in the LAS stability assay are shown below.D130A/V187T, N011K/D027S, A018K/D027S/N073R, D027N/N233Q/P256T,D027S/L075Q/G091Q, E045F/L075D/T189D, G023K/V187T/L264R,I090F/N233Q/P256T, N033D/E045F/L075D, N033D/N073R/T189D,Q004D/S058M/N233Q, A018K/L075Q/G156W/T189D, A018K/L075Q/V187T/T189D,A049V/L075Q/V077I/T189D, D027S/E056K/D111A/V154I,D027S/N033D/L075D/T189D, D027S/N033D/N073R/L075D,D027S/P029E/E045F/L075D, D027S/P029E/N033D/N073R,D048Q/D137Q/G163P/L227M, G023K/D027S/L075Q/L264R,G023K/E056K/L075R/L264R, G023Q/A049V/L075Q/V077I,G023Q/D027S/D111A/G156W, K024A/L075Q/D130A/V154I,N011K/D027Q/S058M/P256T, N011K/E056K/N233Q/P256T,N011K/G023K/L075Q/D111A, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/N011K/D027N/P256T,Q004D/N011K/D027S/P256T, D027E/D048Q/D137Q/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, D027N/S058M/I090F/N233Q/P256T,D027S/P029E/N033D/N073R/L075D, E056K/D130A/V187H/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023Q/D027S/L075G/I252Q/L264R,G023Q/K024A/L075Q/D130A/G156W, L075R/D111A/D130A/V187N/T189Q,N011K/D027N/E056K/N233Q/P256T, N011K/D027N/E056K/S058M/P256T,N011K/D027N/S058M/N233Q/P256T, N011K/D027Q/E056K/S058M/N233Q,N011K/D027Q/I090F/N233Q/P256T, N011K/D027Q/S058M/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/G023K/E056K/L075Q/T189Q,Q004D/D027S/E056K/N233Q/P256T, Q004D/N011K/D027Q/S058M/N233Q,A018K/D027S/P029E/N033D/L075D/T189D,A018K/G023K/D111A/D130A/V154I/T189Q,D027Q/E056K/S058M/D130A/I252Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/G091Q/D130A/V187N/I252Q/L264R,D027S/L075Q/D111A/G156W/V187N/T189Q,D048Q/D130A/G163P/L227M/N233Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/F051T/L075Q/V187T/I252Q,G023Q/D027S/L075R/D111A/V187N/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,N011K/D027N/E056K/S058M/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027Q/S058M/I090F/N233Q/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,N011K/G023K/D027S/V154I/G156W/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,P029E/N033D/K074S/L075D/N101D/T189D,Q004D/D027S/E056K/S058M/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/L075Q/N094R/V154I/G156W/T189Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R

TABLE 6-5 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 in the Detergentstability assay compared to Reference sequence SEQ ID NO: 2 are shownbelow. D130A/V187T, D027S/L075Q/G091Q, G023K/V187T/L264R,I090F/N233Q/P256T, Q004D/S058M/N233Q, D027S/N033D/L075D/T189D,D048Q/D137Q/G163P/L227M, G023K/E056K/L075R/L264R,N011K/D027Q/S058M/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/N011K/D027S/P256T,D027E/D048Q/D137Q/L227M/L264R, D027E/G163P/L227M/N233Q/L264R,E056K/L075R/D130A/V187T/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/D111A/D130A/V187T, G023Q/D027S/L075G/I252Q/L264R,N011K/D027N/S058M/N233Q/P256T, D027Q/E056K/S058M/D130A/I252Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q

TABLE 6-6 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value in the LAS stability assay compared to Reference sequence SEQID NO: 2 are shown below. D130A/V187T, G023K/V187T/L264R,I090F/N233Q/P256T, Q004D/S058M/N233Q, D048Q/D137Q/G163P/L227M,G023K/E056K/L075R/L264R, Q004D/D027Q/N233Q/P256T,Q004D/N011K/D027S/P256T, D027E/D048Q/D137Q/L227M/L264R,D027E/G163P/L227M/N233Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075R/V187N/L264R, G023K/K024A/D111A/D130A/V187T,G023Q/D027S/L075G/I252Q/L264R, N011K/D027N/S058M/N233Q/P256T,D027Q/E056K/S058M/D130A/I252Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,G023Q/E056K/L075Q/D130A/V154I/L264R,Q004D/N011K/D027S/S058M/N233Q/P256T,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q

Example 7 Fabric Adhesion of TLL Combinatorial Variants

The adherence of the TLL variants created as described in Example 2 tocotton fabric in a detergent solution was assayed as described inExample 1(Adhesion Assay). The performance index for increased ordecreased adhesion was calculated for the variants compared to TLL, SEQID NO:1 or the reference sequence SEQ ID NO: 2.

TABLE 7-1 TLL variants with Performance Index >1 in the Fabric Adhesionassay compared to TLL SEQ ID NO: 1 are shown below. A018K/L075Q,D111A/L264R, D130A/L264R, D130A/V187N, D130A/V187T, G023K/L264R,L075R/L264R, S058M/L264R, V187T/L264R, A018K/A049V/V187T,A018K/G023Q/L075Q, A018K/K024A/V154I, A018K/L075Q/G156W,A018K/N033D/T189D, A018K/P029E/T189D, D111A/D130A/L264R,D130A/V187N/L264R, D130A/V187T/L264R, F051T/I252Q/L264R,G023K/D130A/L264R, G023K/D130A/V187T, G023K/E056K/V187T,G023K/L075R/L264R, G023K/V187N/L264R, G023K/V187T/L264R,G023Q/A049V/T189D, G023Q/L075Q/V187T, G163P/L227M/L264R,K024A/L075Q/V077I, L075Q/G156W/T189D, L075Q/G156W/V187N,L075Q/G156W/V187T, L075Q/V154I/V187T, L075Q/V187N/L264R,L075Q/V187T/T189D, L075R/D130A/L264R, L075R/D130A/V187T,N073R/L075D/T189D, P029E/L075D/T189D, A018K/D027S/P029E/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/V187T,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/K024A/D130A/G156W,D027E/D130A/N233Q/L264R, D027E/G163P/L227M/L264R,D027E/S058M/G163P/L264R, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D027S/P029E/L075D/T189D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,D111A/D130A/V154I/G156W, D111A/D130A/V187T/L264R,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023K/L075Q/D130A/V187N,G023K/L075R/V187T/L264R, G023Q/A049V/L075Q/T189D,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/G156W/V187N,G023Q/L075Q/V187T/L264R, G091Q/V187T/I252Q/L264R,K024A/D130A/V154I/V187T, K024A/L075R/G156W/V187N,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,L075Q/D130A/G156W/V187N, L075Q/V077I/D130A/V187Q,L075Q/V077I/V154I/V187Q, L075R/D111A/V154I/G156W,L075R/D130A/V154I/L264R, P029E/N033D/L075D/T189D,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/I252Q/L264R,A018K/D130A/G156W/V187N/L264R, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/L075Q/G156W/V187T,A018K/K024A/L075R/N094R/D130A, A018K/K024A/N094R/D130A/V187N,A018K/K024A/N094R/G156W/V187T, A018K/L075Q/D111A/D130A/V187T,D027E/D048Q/D137Q/L227M/L264R, D027E/D130A/G163P/L227M/L264R,D027E/D130A/G163P/N233Q/L264R, D027E/D137Q/G163P/N233Q/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D130A/G163P/L264R,D027E/S058M/G163P/L227M/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D027S/P029E/N073R/L075D/T189D,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/L075G/V187N/I252Q/L264R, E056K/L075Q/G156W/T189Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075G/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/L264R, F051T/L075Q/V187N/I252Q/L264R,F051T/L075R/V187N/I252Q/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023Q/E056K/V187N/I252Q/L264R,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/K024A/L075Q/G156W/V187N,G023Q/K024A/L075R/G156W/V187Q, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,G023Q/L075Q/V077I/G156W/V187N, G091Q/D130A/V187H/I252Q/L264R,K024A/L075Q/D111A/G156W/V187T, K024A/L075Q/V077I/G156W/V187N,K024A/L075Q/V077I/V154I/V187N, K024A/L075R/V154I/G156W/V187Q,L075G/D130A/V187T/I252Q/L264R, L075Q/D111A/V187N/T189Q/L264R,L075Q/V154I/V187N/T189Q/L264R, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, N011K/G023K/D111A/G156W/L264R,S058M/D130A/G163P/L227M/L264R, A018K/A049V/L075Q/G156W/V187T/T189D,A018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/G156W/V187N/L264R,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/L075Q/V077I/G156W/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/E056K/L075Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,F051T/L075R/D130A/V187N/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,K024A/L075Q/V154I/V187N/T189Q/L264R,K024A/L075Q/V154I/V187T/T189Q/L264R,K024A/N094R/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/V154I/V187N/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/G156W,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D111A/G156W/V187N/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/N094R/D130A/G156W/V187T/L264R,A018K/L075Q/D111A/V154I/V187T/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D111A/V154I/G156W/V187N,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/V187N/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075R/D111A/V154I/V187T/L264R,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075R/V154I/G156W/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V187T/T189Q/L264K,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 7-2 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 in the Fabric Adhesion assay compared to TLL SEQ IDNO: 1 are shown below. D111A/L264R, D130A/L264R, D130A/V187N,D130A/V187T, G023K/L264R, L075R/L264R, S058M/L264R, V187T/L264R,A018K/A049V/V187T, A018K/K024A/V154I, A018K/N033D/T189D,A018K/P029E/T189D, D111A/D130A/L264R, D130A/V187N/L264R,D130A/V187T/L264R, F051T/I252Q/L264R, G023K/D130A/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187N/L264R, G023K/V187T/L264R, G023Q/A049V/T189D,G163P/L227M/L264R, L075Q/V187N/L264R, L075R/D130A/L264R,L075R/D130A/V187T, A018K/D027S/P029E/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/V187T, A018K/K024A/D130A/G156W,D027E/D130A/N233Q/L264R, D027E/G163P/L227M/L264R,D027E/S058M/G163P/L264R, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D027S/P029E/L075D/T189D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,D111A/D130A/V154I/G156W, D111A/D130A/V187T/L264R,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023K/L075R/V187T/L264R,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, S058M/L075Q/I252Q/L264R,A018K/D130A/G156W/V187N/L264R, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/K024A/N094R/D130A/V187N,A018K/K024A/N094R/G156W/V187T, D027E/D048Q/D137Q/L227M/L264R,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/G163P/L227M/L264R,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075R/V154I/T189Q/L264R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/L075G/V187N/I252Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023Q/E056K/V187N/I252Q/L264R,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,N011K/G023K/D111A/G156W/L264R, S058M/D130A/G163P/L227M/L264R,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/K024A/V154I/G156W/V187Q,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 7-3 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value in the Fabric Adhesionassay compared to TLL SEQ ID NO: 1 are shown below. D111A/L264R,D130A/L264R, D130A/V187N, D130A/V187T, G023K/L264R, L075R/L264R,S058M/L264R, V187T/L264R, A018K/A049V/V187T, A018K/K024A/V154I,D111A/D130A/L264R, D130A/V187N/L264R, D130A/V187T/L264R,F051T/I252Q/L264R, G023K/D130A/L264R, G023K/E056K/V187T,G023K/L075R/L264R, G023K/V187T/L264R, L075Q/V187N/L264R,L075R/D130A/L264R, A018K/G023K/D111A/T189Q, D027E/D130A/N233Q/L264R,D027S/D130A/I252Q/L264R, D048Q/D130A/G163P/L264R,D111A/D130A/V187T/L264R, E056K/D130A/V187N/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/L075R/L264R, G023K/K024A/V154I/V187N,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, S058M/G163P/L227M/L264R,S058M/L075Q/I252Q/L264R, A018K/D130A/G156W/V187N/L264R,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/K024A/N094R/D130A/V187N,D027E/D048Q/D137Q/L227M/L264R, D027E/D130A/G163P/L227M/L264R,D027E/D130A/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/D137Q/G163P/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/L075G/V187N/I252Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/L075R/D130A/V187N/L264R, G023Q/E056K/V187N/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,L075G/D130A/V187T/I252Q/L264R, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 7-4 TLL variants with Performance Index >1 in the Fabric Adhesionassay compared to Reference sequence SEQ ID NO: 2 are shown below.D111A/L264R, D130A/L264R, D130A/V187N, D130A/V187T, G023K/L264R,L075R/L264R, S058M/L264R, V187T/L264R, A018K/A049V/V187T,A018K/K024A/V154I, A018K/N033D/T189D, D111A/D130A/L264R,D130A/V187N/L264R, D130A/V187T/L264R, F051T/I252Q/L264R,G023K/D130A/L264R, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187T/L264R, G023Q/L075Q/V187T, L075Q/G156W/V187N,L075Q/G156W/V187T, L075Q/V187N/L264R, L075R/D130A/L264R,A018K/D027S/P029E/T189D, A018K/G023K/D111A/T189Q,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, D027E/D130A/N233Q/L264R,D027E/G163P/L227M/L264R, D027S/D130A/I252Q/L264R,D048Q/D130A/G163P/L264R, D111A/D130A/V154I/G156W,D111A/D130A/V187T/L264R, E056K/D130A/V187N/L264R,E056K/D130A/V187T/L264R, F051T/L075G/I252Q/L264R,F051T/L075Q/I252Q/L264R, G023K/D130A/V187N/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/L075R/L264R,G023K/K024A/V154I/V187N, G023K/L075Q/D130A/V187N,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,K024A/L075R/G156W/V187N, L075G/D130A/V187T/I252Q,L075G/V187H/I252Q/L264R, L075Q/V077I/D130A/V187Q,P029E/N033D/L075D/T189D, S058M/G163P/L227M/L264R,S058M/L075Q/I252Q/L264R, A018K/D130A/G156W/V187N/L264R,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/L075Q/G156W/V187T, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, D027E/D048Q/D137Q/L227M/L264R,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/G163P/L227M/N233Q/L264R, D027E/S058M/D130A/G163P/L264R,D027S/F051T/V187N/I252Q/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075R/V154I/T189Q/L264R,D027S/P029E/N073R/L075D/T189D, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/D137Q/G163P/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/L075G/V187N/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, F051T/D130A/V187T/I252Q/L264R,F051T/L075G/D130A/I252Q/L264R, F051T/L075G/G091Q/D130A/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/L075Q/D130A/G156W/L264R, G023K/L075R/D130A/V187N/L264R,G023Q/E056K/V187N/I252Q/L264R, G023Q/K024A/L075Q/G156W/V187N,G023Q/K024A/L075R/G156W/V187Q, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,G023Q/L075Q/V077I/G156W/V187N, G091Q/D130A/V187H/I252Q/L264R,K024A/L075Q/D111A/G156W/V187T, L075G/D130A/V187T/I252Q/L264R,L075Q/D111A/V187N/T189Q/L264R, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, S058M/D130A/G163P/L227M/L264R,A018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/G156W/V187N/L264R,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/G156W/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/E056K/L075Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,F051T/L075R/D130A/V187N/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,G023Q/L075R/V154I/G156W/V187N/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D111A/G156W/V187N/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/N094R/D130A/G156W/V187T/L264R,A018K/L075Q/D111A/V154I/V187T/T189Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D111A/V154I/G156W/V187N,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/E056K/L075Q/D111A/G156W/V187N/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/V187N/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075R/D111A/V154I/V187T/L264R,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075R/V154I/G156W/V187N/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 7-5 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 in the FabricAdhesion assay compared to Reference sequence SEQ ID NO: 2 are shownbelow. D111A/L264R, D130A/L264R, D130A/V187N, D130A/V187T, G023K/L264R,L075R/L264R, S058M/L264R, V187T/L264R, A018K/A049V/V187T,A018K/K024A/V154I, A018K/N033D/T189D, D111A/D130A/L264R,D130A/V187N/L264R, D130A/V187T/L264R, F051T/I252Q/L264R,G023K/D130A/L264R, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187T/L264R, L075Q/V187N/L264R, L075R/D130A/L264R,A018K/D027S/P029E/T189D, A018K/G023K/D111A/T189Q,D027E/D130A/N233Q/L264R, D027E/G163P/L227M/L264R,D027S/D130A/I252Q/L264R, D048Q/D130A/G163P/L264R,D111A/D130A/V154I/G156W, D111A/D130A/V187T/L264R,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/L075R/L264R, G023K/K024A/V154I/V187N,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,S058M/G163P/L227M/L264R, S058M/L075Q/I252Q/L264R,A018K/D130A/G156W/V187N/L264R, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/K024A/N094R/D130A/V187N,D027E/D048Q/D137Q/L227M/L264R, D027E/D130A/G163P/L227M/L264R,D027E/D130A/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/D137Q/G163P/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/L075G/V187N/I252Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075G/V187N/I252Q/L264R,F051T/L075Q/V187N/I252Q/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/L075R/D130A/V187N/L264R,G023Q/E056K/V187N/I252Q/L264R, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,L075G/D130A/V187T/I252Q/L264R, S058M/D130A/G163P/L227M/L264R,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 7-6 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value in the Fabric Adhesion assay compared to Reference sequence SEQID NO: 2 are shown below. D111A/L264R, D130A/L264R, D130A/V187N,D130A/V187T, G023K/L264R, L075R/L264R, S058M/L264R, V187T/L264R,A018K/A049V/V187T, A018K/K024A/V154I, D111A/D130A/L264R,D130A/V187N/L264R, D130A/V187T/L264R, F051T/I252Q/L264R,G023K/D130A/L264R, G023K/E056K/V187T, G023K/L075R/L264R,G023K/V187T/L264R, L075Q/V187N/L264R, L075R/D130A/L264R,A018K/G023K/D111A/T189Q, D027E/D130A/N233Q/L264R,D027S/D130A/I252Q/L264R, D048Q/D130A/G163P/L264R,D111A/D130A/V187T/L264R, E056K/D130A/V187N/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/L075R/L264R, G023K/K024A/V154I/V187N,G023Q/F051T/I252Q/L264R, G023Q/G091Q/I252Q/L264R,G023Q/L075Q/D130A/L264R, G023Q/L075Q/V187T/L264R,G091Q/V187T/I252Q/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, S058M/G163P/L227M/L264R,S058M/L075Q/I252Q/L264R, A018K/D130A/G156W/V187N/L264R,A018K/G023K/K024A/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/K024A/N094R/D130A/V187N,D027E/D048Q/D137Q/L227M/L264R, D027E/D130A/G163P/L227M/L264R,D027E/D130A/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027S/F051T/V187N/I252Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075G/G091Q/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/S058M/D137Q/N233Q/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/D137Q/G163P/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/L075G/V187N/I252Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/V187N/I252Q/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/L075R/D130A/V187N/L264R, G023Q/E056K/V187N/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/G091Q/I252Q/L264R,L075G/D130A/V187T/I252Q/L264R, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/V187H/I252Q/L264R,F051T/L075G/G091Q/V187T/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023Q/D027S/F051T/L075Q/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/G091Q/V187N/I252Q/L264R,K024A/L075Q/D111A/V154I/V187N/T189Q,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075Q/V187N/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/L075Q/V187H/I252Q/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075G/G091Q/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R

TABLE 7-7 TLL variants with Performance Index <1 in the Fabric Adhesionassay compared to TLL SEQ ID NO: 1 are shown below. A018K/D027S,A018K/E045F, A018K/L075D, A018K/T189D, D027E/G163P, D027N/E056K,D027N/N233Q, D027S/N033D, D027S/P256T, D130A/T189Q, E045F/N073R,E045F/T189D, F051T/L075R, G023K/D130A, G023Q/L075Q, K024A/L075Q,K024A/V154I, L075Q/D130A, L075Q/G156W, L227M/L264R, N011K/D027S,N033D/E045F, N073R/L075D, N094R/G156W, P029E/N033D, A018K/A049V/L075Q,A018K/D027S/E045F, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/N073R, A018K/E045F/T189D, A018K/E045F/V187T,A018K/G023K/G156W, A018K/G023K/L075Q, A018K/L075D/T189D,A018K/L075Q/V187T, A018K/N033D/L075D, A018K/N073R/L075D,A018K/V154I/G156W, A049V/L075Q/T189D, A049V/V187T/T189D,D027E/S058M/G163P, D027N/N233Q/P256T, D027N/S058M/P256T,D027Q/N233Q/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N033D/T189D, D027S/N073R/L075D, D027S/N233Q/P256T,D027S/S058M/P256T, D130A/T189Q/L264R, D130A/V154I/G156W,E045F/L075D/T189D, E045F/N073R/L075D, E056K/D130A/L264R,E056K/D130A/T189Q, G023Q/D111A/L264R, G023Q/D130A/G156W,G023Q/E045F/V077I, G023Q/E056K/L075R, G023Q/K024A/G156W,G023Q/L075Q/V077I, I090F/N233Q/P256T, K024A/D130A/V154I,K024A/L075Q/G156W, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/D130A/V187T, L075Q/N094R/V154I, L075Q/V077I/T189D,L075Q/V187T/L264R, L075R/V187N/L264R, L075R/V187T/L264R,N011K/D027S/S058M, N011K/N233Q/P256T, N033D/E045F/L075D,N033D/E045F/N073R, N033D/N073R/T189D, N094R/D130A/V187T,P029E/E045F/L075D, P029E/N033D/E045F, P029E/N033D/L075D,P029E/N073R/L075D, Q004D/D027S/P256T, Q004D/N233Q/P256T,Q004D/S058M/N233Q, S058M/L227M/L264R, V077I/D130A/V154I,V077I/V187A/T189D, V187N/T189Q/L264R, A018K/A049V/L075Q/T189D,A018K/A049V/L075Q/V187T, A018K/D027S/E045F/N073R,A018K/D027S/E045F/T189D, A018K/D027S/L075D/T189D,A018K/D027S/N033D/L075D, A018K/D111A/G156W/T189Q,A018K/D130A/G156W/V187T, A018K/E045F/A049V/G156W,A018K/E045F/A049V/V187T, A018K/E045F/L075D/T189D,A018K/E045F/L075Q/T189D, A018K/E045F/L075Q/V187T,A018K/G023Q/A049V/T189D, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/G156W/V187T,A018K/G023Q/L075Q/V077I, A018K/G023Q/V077I/G156W,A018K/G023Q/V077I/V187T, A018K/G023Q/V187T/T189D,A018K/L075Q/G156W/T189D, A018K/L075Q/G156W/V187T,A018K/L075Q/N094R/D111A, A018K/L075Q/N094R/D130A,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/L075Q/V187T/T189D, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/L075D,A018K/P029E/N033D/T189D, A018K/P029E/N073R/L075D,A018K/V154I/G156W/V187T, A049V/L075Q/V077I/T189D,A049V/L075Q/V187T/T189D, D027E/D048Q/G163P/L264R,D027E/D137Q/G163P/L227M, D027E/D137Q/L227M/L264R,D027E/L227M/N233Q/L264R, D027N/E056K/N233Q/P256T,D027N/E056K/S058M/P256T, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027Q/S058M/I090F/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/L075D,D027S/E045F/N073R/T189D, D027S/E056K/D111A/V154I,D027S/E056K/I090F/P256T, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/N033D/N073R/L075D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/L075D,D027S/P029E/E045F/N073R, D027S/P029E/E045F/T189D,D027S/P029E/N033D/E045F, 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G023Q/K024A/L075Q/V077I/V187Q,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/V077I/D130A,G023Q/K024A/V077I/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/D111A/V154I/V187T, K024A/L075R/G156W/V187N/T189Q,L075Q/D111A/D130A/V187T/T189Q, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075Q/V077I/D130A/G156W/V187N,L075R/D111A/D130A/V187N/T189Q, L075R/G156W/V187T/T189Q/L264R,L075R/V077I/N094R/V154I/G156W, N011K/D027N/E056K/N233Q/P256T,N011K/D027N/E056K/S058M/P256T, N011K/D027N/S058M/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q, N011K/D027Q/I090F/N233Q/P256T,N011K/D027Q/S058M/I090F/P256T, N011K/D027Q/S058M/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023K/E056K/L075Q/T189Q, N011K/G023Q/L075Q/V187N/T189Q,P029E/N033D/E045F/L075D/T189D, P029E/N033D/K074S/L075D/T189D,P029E/N033D/N073R/L075D/T189D, Q004D/D027N/E056K/S058M/P256T,Q004D/D027N/I090F/N233Q/P256T, Q004D/D027Q/E056K/N233Q/P256T,Q004D/D027Q/E056K/S058M/P256T, Q004D/D027S/E056K/N233Q/P256T,Q004D/D027S/E056K/S058M/P256T, Q004D/D027S/S058M/N233Q/P256T,Q004D/N011K/D027N/N233Q/P256T, Q004D/N011K/D027Q/E056K/N233Q,Q004D/N011K/D027Q/S058M/N233Q, Q004D/N011K/I090F/N233Q/P256T,S058M/D130A/D137Q/G163P/L264R, S058M/D137Q/G163P/L227M/L264R,S058M/D137Q/G163P/L227M/N233Q, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/L075D/T189D,A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/D027S/P029E/N033D/L075D/T189D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/E045F/A049V/L075Q/V077I/V187T,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/D111A/L264R,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/A049V/L075Q/V077I/G156W,A018K/G023Q/A049V/L075Q/V077I/V187T,A018K/G023Q/A049V/V077I/V187T/T189D,A018K/G023Q/E045F/A049V/G156W/V187T,A018K/G023Q/E045F/A049V/L075Q/T189D,A018K/G023Q/E045F/A049V/L075Q/V077I,A018K/G023Q/E045F/A049V/L075Q/V187T,A018K/G023Q/E045F/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156C/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/G023Q/L075Q/V077I/V187T/T189D,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/V154I/G156W/V187N,A018K/L075Q/D111A/V154I/T189Q/L264R,A018K/N033D/E045F/N073R/L075D/T189D,A018K/P029E/N033D/E045F/L075D/T189D,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/E056K/S058M/I090F/N233Q/P256T,D027Q/F051T/L075G/D130A/I252Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/G091Q/D130A/V187N/I252Q/L264R,D027S/L075Q/D111A/G156W/V187N/T189Q,D027S/P029E/N033D/E045F/N073R/L075D,D027S/P029E/N033D/E045F/N073R/T189D,D027S/P029E/N033D/N073R/L075D/T189D,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,E045F/A049V/L075Q/V077I/V187T/T189D,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/G156W/V187T/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/A049V/L075Q/G156W/V187T/T189D,G023Q/D027Q/E056K/L075R/I252Q/L264R,G023Q/D027S/F051T/L075Q/V187T/I252Q,G023Q/D027S/L075Q/D111A/G156W/L264R,G023Q/D027S/L075R/D111A/V187N/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E045F/A049V/L075Q/G156W/V187T,G023Q/E045F/A049V/L075Q/V077I/V187T,G023Q/E045F/L075Q/V077I/G156W/V187T,G023Q/E045F/L075Q/V077I/V187T/T189D,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/L075Q/V154I/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/D111A/V154I/T189Q/L264R,G023Q/L075Q/N094R/D111A/G156W/L264R,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,K024A/L075Q/V154I/G156W/T189Q/L264R,K024A/L075R/D111A/V154I/G156W/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/N094R/D111A/G156W/T189Q/L264R,N011K/D027N/E056K/I090F/N233Q/P256T,N011K/D027N/E056K/S058M/N233Q/P256T,N011K/D027N/S058M/I090F/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027Q/S058M/I090F/N233Q/P256T,N011K/D027S/E056K/I090F/N233Q/P256T,N011K/D027S/E056K/S058M/I090F/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,N011K/D027S/S058M/I090F/N233Q/P256T,N011K/G023K/D027S/V154I/G156W/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/N094R/D130A/L264R,P029E/N033D/K074S/L075D/N101D/T189D,Q004D/D027N/E056K/S058M/N233Q/P256T,Q004D/D027S/E056K/S058M/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q,Q004D/N011K/D027S/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/E045F/A049V/L075Q/G156W/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/N094R/G156W/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/D130A/G156W/V187N/T189Q/L264R,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027S/E056K/L075R/D111A/G156W/V187N/L264R,D027S/L075R/V154I/G156W/V187N/T189Q/L264R,D027S/P029E/N033D/E045F/N073R/L075D/T189D,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/D027S/L075Q/N094R/V154I/G156W/T189Q,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/D111A/V154I/G156W/T189Q/L264R,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/V187N/L264R,G023K/L075Q/N094R/V154I/G156W/V187T/L264R,G023Q/D027S/L075Q/V154I/G156W/V187N/L264R,G023Q/E045F/A049V/V077I/G156W/V187T/T189D,G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/E056K/L075R/G091Q/V187H/I252Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/D027N/E056K/S058M/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,Q004D/N011K/D027Q/S058M/I090F/N233Q/P256T,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187T/L264R,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075Q/D111A/V154I/V187N/T189Q/L264R,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023K/L075Q/D111A/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 7-8 TLL variants with Performance Index for expression ≥0.5 andPerformance Index <1 in the Fabric Adhesion assay compared to TLL SEQ IDNO: 1 are shown below. A018K/E045F, A018K/L075D, D027E/G163P,D027S/N033D, D027S/P256T, E045F/N073R, F051T/L075R, K024A/V154I,L227M/L264R, N033D/E045F, N094R/G156W, P029E/N033D, A018K/E045F/N073R,A018K/E045F/V187T, A018K/L075D/T189D, A018K/V154I/G156W,D027E/S058M/G163P, D027N/S058M/P256T, D027Q/S058M/P256T,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/S058M/P256T,D130A/V154I/G156W, E056K/D130A/L264R, G023Q/D130A/G156W,G023Q/K024A/G156W, I090F/N233Q/P256T, K024A/D130A/V154I,L075G/D130A/V187H, L075Q/V187T/L264R, L075R/V187N/L264R,L075R/V187T/L264R, N011K/N233Q/P256T, N094R/D130A/V187T,P029E/N033D/E045F, Q004D/D027S/P256T, Q004D/S058M/N233Q,V187N/T189Q/L264R, A018K/D111A/G156W/T189Q, A018K/D130A/G156W/V187T,A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/G023Q/V077I/G156W, A018K/L075QA/V077I/N094R,A018K/N033D/L075D/T189D, A018K/P029E/N073R/L075D,D027E/D048Q/G163P/L264R, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/L227M/N233Q/L264R,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/N073R,D027S/P029E/E045F/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D137Q/G163P/L227M,D130A/G163P/L227M/L264R, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, F051T/D130A/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/E056K/D130A/V187N, G023K/E056K/V187T/L264R,G023K/L075R/D130A/L264R, G023Q/A049V/V077I/G156W,G023Q/K024A/D130A/G156W, G023Q/K024A/L075R/G156W,G023Q/K024A/V077I/G156W, L075G/G091Q/V187N/L264R,L075R/D130A/V187T/L264R, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/N073R/L075D/T189D, Q004D/D027N/E056K/P256T,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/N011K/D027S/P256T,S058M/D137Q/G163P/N233Q, A018K/D027S/E045F/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/E045F/N073R/L075D/T189D,A018K/G023K/D130A/V154I/G156W, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/V077I/D130A/G156W, A018K/K024A/L075R/D130A/V154I,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L264R, D027E/S058M/D137Q/G163P/N233Q,D027Q/F051T/L075Q/D130A/L264R, D027S/E056K/D111A/V187N/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D048Q/S058M/G163P/L227M/L264R,D048Q/S058M/G163P/N233Q/L264R, E045F/A049V/L075Q/V187T/T189D,E056K/L075R/D130A/V187N/L264R, E056K/L075R/D130A/V187T/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/K024A/D111A/D130A/V187T, G023K/K024A/D111A/G156W/T189Q,G023K/L075Q/V187H/I252Q/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/D027S/L075G/I252Q/L264R, G023Q/K024A/L075Q/G156W/V187Q,G023Q/L075Q/V077I/D130A/G156W, N011K/D027N/S058M/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, Q004D/N011K/D027N/N233Q/P256T,S058M/D130A/D137Q/G163P/L264R, S058M/D137Q/G163P/L227M/L264R,S058M/D137Q/G163P/L227M/N233Q, S058M/G163P/L227M/N233Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023Q/K024A/L075R/N094R/G156W,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027E/D048Q/S058M/G163P/L227M/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023K/K024A/N094R/V154I/G156W/V187N/T189Q,G023Q/F051T/E056K/L075R/G091Q/V187H/I252Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 7-9 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≤50% of the maximum PI value in the Fabric Adhesionassay compared to TLL SEQ ID NO: 1 are shown below. A018K/E045F,A018K/L075D, D027E/G163P, D027S/P256T, E045F/N073R, F051T/L075R,K024A/V154I, L227M/L264R, N033D/E045F, A018K/E045F/N073R,A018K/E045F/V187T, A018K/V154I/G156W, D027N/S058M/P256T,D027Q/S058M/P256T, D027S/L075Q/G091Q, D027S/S058M/P256T,E056K/D130A/L264R, G023Q/K024A/G156W, I090F/N233Q/P256T,N011K/N233Q/P256T, N094R/D130A/V187T, P029E/N033D/E045F,Q004D/D027S/P256T, Q004D/S058M/N233Q, A018K/D111A/G156W/T189Q,A018K/E045F/A049V/G156W, A018K/E045F/L075D/T189D,A018K/P029E/N073R/L075D, D027E/D137Q/L227M/L264R,D027N/S058M/N233Q/P256T, D027Q/E056K/N233Q/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/T189D,D027S/N033D/E045F/N073R, D027S/N033D/L075D/T189D,D027S/P029E/E045F/N073R, D027S/P029E/E045F/T189D,D027S/P029E/N033D/E045F, D048Q/D137Q/G163P/L227M,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,G023K/E056K/D130A/V187N, G023K/E056K/V187T/L264R,G023Q/K024A/L075R/G156W, L075G/G091Q/V187N/L264R,N011K/D027Q/S058M/P256T, N011K/D027S/N233Q/P256T,N033D/E045F/N073R/T189A, Q004D/D027N/E056K/P256T,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/N011K/D027S/P256T,A018K/D027S/E045F/L075D/T189D, A018K/E045F/N073R/L075D/T189D,A018K/K024A/L075R/D130A/V154I, D027E/D130A/D137Q/G163P/L264R,D027Q/F051T/L075Q/D130A/L264R, D027S/E056K/D111A/V187N/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/G163P/L227M/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/L075R/D130A/V187N/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/L075R/V187N/L264R,G023K/K024A/D111A/D130A/V187T, G023K/L075Q/V187H/I252Q/L264R,G023Q/D027S/L075G/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,N011K/D027N/S058M/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,Q004D/N011K/D027N/N233Q/P256T, S058M/D137Q/G163P/L227M/N233Q,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027Q/E056K/S058M/D130A/I252Q/L264R,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,G023Q/F051T/E056K/L075R/G091Q/V187H/I252Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 7-10 TLL variants with Performance Index <1 in the Fabric Adhesionassay compared to Reference sequence SEQ ID NO: 2 are shown below.A018K/D027S, A018K/E045F, A018K/L075D, A018K/T189D, D027E/G163P,D027N/E056K, D027N/N233Q, D027S/P256T, D130A/T189Q, E045F/N073R,E045F/T189D, F051T/L075R, G023K/D130A, G023Q/L075Q, K024A/V154I,L227M/L264R, N011K/D027S, N033D/E045F, N073R/L075D, A018K/A049V/L075Q,A018K/D027S/E045F, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/N073R, A018K/E045F/T189D, A018K/E045F/V187T,A018K/G023K/G156W, A018K/G023K/L075Q, A018K/N033D/L075D,A018K/N073R/L075D, A018K/V154I/G156W, A049V/L075Q/T189D,A049V/V187T/T189D, D027N/N233Q/P256T, D027N/S058M/P256T,D027Q/N233Q/P256T, D027Q/S058M/P256T, D027S/L075Q/G091Q,D027S/N073R/L075D, D027S/N233Q/P256T, D027S/S058M/P256T,D130A/V154I/G156W, E045F/L075D/T189D, E045F/N073R/L075D,E056K/D130A/L264R, E056K/D130A/T189Q, G023Q/D111A/L264R,G023Q/E045F/V077I, G023Q/E056K/L075R, G023Q/K024A/G156W,G023Q/L075Q/V077I, I090F/N233Q/P256T, K024A/L075Q/G156W,L075Q/D130A/V187T, L075Q/N094R/V154I, L075Q/V077I/T189D,N011K/D027S/S058M, N011K/N233Q/P256T, N033D/E045F/L075D,N033D/E045F/N073R, N033D/N073R/T189D, N094R/D130A/V187T,P029E/E045F/L075D, P029E/N033D/E045F, P029E/N033D/L075D,P029E/N073R/L075D, Q004D/D027S/P256T, Q004D/N233Q/P256T,Q004D/S058M/N233Q, S058M/L227M/L264R, V077I/D130A/V154I,V077I/V187A/T189D, A018K/A049V/L075Q/T189D, A018K/D027S/E045F/N073R,A018K/D027S/E045F/T189D, A018K/D027S/L075D/T189D,A018K/D027S/N033D/L075D, A018K/D111A/G156W/T189Q,A018K/E045F/A049V/G156W, A018K/E045F/A049V/V187T,A018K/E045F/L075D/T189D, A018K/E045F/L075Q/T189D,A018K/E045F/L075Q/V187T, A018K/G023Q/A049V/T189D,A018K/G023Q/E045F/T189D, A018K/G023Q/G156W/V187T,A018K/G023Q/V077I/V187T, A018K/L075Q/G156W/T189D,A018K/L075Q/G156W/V187T, A018K/L075Q/N094R/D111A,A018K/L075Q/N094R/D130A, A018K/L075Q/V187T/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/T189D,A018K/P029E/N073R/L075D, A018K/V154I/G156W/V187T,A049V/L075Q/V077I/T189D, A049V/L075Q/V187T/T189D,D027E/D137Q/L227M/L264R, D027N/E056K/N233Q/P256T,D027N/E056K/S058M/P256T, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027Q/S058M/I090F/P256T,D027S/E045F/L075D/T189D, D027S/E045F/N073R/L075D,D027S/E045F/N073R/T189D, D027S/E056K/D111A/V154I,D027S/E056K/I090F/P256T, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/N033D/N073R/L075D,D027S/P029E/E045F/L075D, D027S/P029E/E045F/N073R,D027S/P029E/E045F/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/N073R, D048Q/D137Q/G163P/L227M,E045F/A049V/G156W/V187T, E045F/L075Q/G156W/V187T,G023K/D027S/L075Q/L264R, G023K/D111A/D130A/L264R,G023K/E056K/D130A/L264R, G023K/E056K/D130A/V187N,G023K/E056K/V187T/L264R, G023K/L075Q/G156W/L264R,G023K/L075R/V187N/L264R, G023Q/A049V/L075Q/V077I,G023Q/D027S/D111A/G156W, G023Q/E045F/A049V/T189D,G023Q/E045F/A049V/V187T, G023Q/E045F/L075Q/G156W,G023Q/E045F/L075Q/V187T, G023Q/K024A/L075R/G156W,G023Q/V154I/G156W/V187N, K024A/L075Q/D130A/G156W,K024A/L075Q/D130A/V154I, L075G/G091Q/V187N/L264R,L075Q/D130A/V154I/G156W, L075Q/G156W/V187T/L264R,L075Q/V077I/G156W/V187N, L075Q/V077I/V187T/T189D,L075Q/V187N/T189Q/L264R, N011K/D027N/E056K/S058M,N011K/D027N/S058M/P256T, N011K/D027Q/S058M/P256T,N011K/D027S/E056K/P256T, N011K/D027S/N233Q/P256T,N011K/D027S/S058M/N233Q, N011K/E056K/N233Q/P256T,N011K/E056K/S058M/P256T, N011K/G023K/L075Q/D111A,N011K/S058M/N233Q/P256T, N033D/E045F/N073R/T189A,P029E/E045F/L075D/T189D, P029E/E045F/N073R/L075D,P029E/E045F/N073R/T189D, P029E/N033D/E045F/L075D,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/D027S/I090F/P256T, Q004D/N011K/D027N/P256T,Q004D/N011K/D027Q/N233Q, Q004D/N011K/D027S/P256T,Q004D/N011K/E056K/S058M, A018K/A049V/L075Q/V187T/T189D,A018K/D027S/E045F/L075D/T189D, A018K/D027S/E045F/N073R/D137V,A018K/D027S/N033D/E045F/T189D, A018K/D027S/N033D/N073R/T189D,A018K/D027S/N073R/L075D/T189D, A018K/D027S/P029E/E045F/T189D,A018K/D027S/P029E/N073R/L075D, A018K/D130A/G156W/V187T/L264R,A018K/E045F/A049V/L075Q/G156W, A018K/E045F/A049V/L075Q/T189D,A018K/E045F/A049V/L075Q/V187T, A018K/E045F/N073R/L075D/T189D,A018K/G023K/L075Q/D130A/V154I, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V077I, A018K/G023Q/A049V/L075Q/V187T,A018K/G023Q/A049V/V077I/V187T, A018K/G023Q/E045F/A049V/L075Q,A018K/G023Q/E045F/A049V/V187T, A018K/G023Q/E045F/L075Q/V187T,A018K/G023Q/E045F/V077I/T189D, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/V154I/T189D,A018K/K024A/L075R/D130A/V154I, A018K/L075Q/G156W/V187T/T189D,A018K/L075Q/V187T/T189Q/L264R, A018K/N033D/E045F/L075D/T189D,A018K/P029E/E045F/N073R/L075D, A018K/V077I/G156W/V187T/T189D,D027E/D048Q/L227M/N233Q/L264R, D027E/D048Q/S058M/G163P/N233Q,D027E/D130A/D137Q/G163P/L264R, D027E/D137Q/G163P/L227M/L264R,D027N/E056K/S058M/N233Q/P256T, D027N/S058M/I090F/N233Q/P256T,D027Q/E056K/I090F/N233Q/P256T, D027Q/E056K/S058M/N233Q/P256T,D027Q/F051T/L075Q/D130A/L264R, D027S/E056K/D111A/V187N/L264R,D027S/E056K/S058M/N233Q/P256T, D027S/L075Q/D111A/D130A/V187N,D027S/N033D/E045F/N073R/L075D, D027S/P029E/E045F/N073R/L075D,D027S/P029E/E045F/N073R/T189D, D027S/P029E/N033D/E045F/N073R,D027S/P029E/N033D/N073R/L075D, D048Q/D130A/D137Q/G163P/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D137Q/G163P/L264R,D048Q/S058M/G163P/L227M/L264R, D130A/G163P/L227M/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/D130A/V187H/I252Q/L264R,E056K/L075R/D130A/V187N/L264R, E056K/N094R/G156W/V187N/L264R,E056K/S058M/I090F/N233Q/P256T, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/D111A/V154I/V187T/L264R,G023K/E056K/L075R/V187N/L264R, G023K/K024A/D111A/D130A/V187T,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D130A/V154I/T189Q,G023K/L075Q/V187H/I252Q/L264R, G023Q/A049V/L075Q/G156W/T189D,G023Q/A049V/L075Q/V187T/T189D, G023Q/D027S/D111A/T189Q/L264R,G023Q/D027S/L075G/I252Q/L264R, G023Q/D027S/V154I/V187N/L264R,G023Q/E045F/A049V/G156W/T189D, G023Q/E045F/A049V/L075Q/V077I,G023Q/E045F/A049V/V077I/T189D, G023Q/E045F/L075Q/G156W/T189D,G023Q/E045F/L075Q/V077I/V187T, G023Q/K024A/L075Q/D130A/G156W,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/V077I/D130A,G023Q/L075Q/V077I/D130A/G156W, K024A/L075R/D111A/V154I/V187T,K024A/L075R/G156W/V187N/T189Q, L075Q/D111A/D130A/V187T/T189Q,L075Q/D130A/V187T/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075R/D111A/D130A/V187N/T189Q,L075R/V077I/N094R/V154I/G156W, N011K/D027N/E056K/N233Q/P256T,N011K/D027N/E056K/S058M/P256T, N011K/D027N/S058M/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q, N011K/D027Q/I090F/N233Q/P256T,N011K/D027Q/S058M/I090F/P256T, N011K/D027Q/S058M/N233Q/P256T,N011K/D027S/E056K/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,N011K/G023Q/L075Q/V187N/T189Q, P029E/N033D/E045F/L075D/T189D,P029E/N033D/K074S/L075D/T189D, Q004D/D027N/E056K/S058M/P256T,Q004D/D027N/I090F/N233Q/P256T, Q004D/D027Q/E056K/N233Q/P256T,Q004D/D027Q/E056K/S058M/P256T, Q004D/D027S/E056K/N233Q/P256T,Q004D/D027S/E056K/S058M/P256T, Q004D/D027S/S058M/N233Q/P256T,Q004D/N011K/D027N/N233Q/P256T, Q004D/N011K/D027Q/E056K/N233Q,Q004D/N011K/D027Q/S058M/N233Q, Q004D/N011K/I090F/N233Q/P256T,S058M/D137Q/G163P/L227M/N233Q, V077I/D130A/V154I/G156W/V187N,A018K/D027S/N033D/E045F/L075D/T189D,A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/D027S/P029E/N033D/L075D/T189D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/E045F/A049V/L075Q/V077I/V187T,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/D111A/L264R,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/A049V/L075Q/V077I/G156W,A018K/G023Q/A049V/V077I/V187T/T189D,A018K/G023Q/E045F/A049V/G156W/V187T,A018K/G023Q/E045F/A049V/L075Q/T189D,A018K/G023Q/E045F/A049V/L075Q/V077I,A018K/G023Q/E045F/A049V/L075Q/V187T,A018K/G023Q/E045F/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/G156C/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/G023Q/L075Q/V077I/N094R/V154I,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/V154I/G156W/V187N,A018K/L075Q/D111A/V154I/T189Q/L264R,A018K/N033D/E045F/N073R/L075D/T189D,A018K/P029E/N033D/E045F/L075D/T189D,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/E056K/S058M/I090F/N233Q/P256T,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/G091Q/D130A/V187N/I252Q/L264R,D027S/L075Q/D111A/G156W/V187N/T189Q,D027S/P029E/N033D/E045F/N073R/L075D,D027S/P029E/N033D/E045F/N073R/T189D,D027S/P029E/N033D/N073R/L075D/T189D,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,E045F/A049V/L075Q/V077I/V187T/T189D,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/G156W/V187T/T189Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/A049V/L075Q/G156W/V187T/T189D,G023Q/D027S/F051T/L075Q/V187T/I252Q,G023Q/D027S/L075Q/D111A/G156W/L264R,G023Q/D027S/L075R/D111A/V187N/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E045F/A049V/L075Q/G156W/V187T,G023Q/E045F/A049V/L075Q/V077I/V187T,G023Q/E045F/L075Q/V077I/G156W/V187T,G023Q/E045F/L075Q/V077I/V187T/T189D,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/N094R/D111A/G156W/L264R,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075R/D111A/V154I/G156W/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/N094R/D111A/G156W/T189Q/L264R,N011K/D027N/E056K/I090F/N233Q/P256T,N011K/D027N/E056K/S058M/N233Q/P256T,N011K/D027N/S058M/I090F/N233Q/P256T,N011K/D027Q/E056K/S058M/N233Q/P256T,N011K/D027Q/S058M/I090F/N233Q/P256T,N011K/D027S/E056K/I090F/N233Q/P256T,N011K/D027S/E056K/S058M/I090F/P256T,N011K/D027S/E056K/S058M/N233Q/P256T,N011K/D027S/S058M/I090F/N233Q/P256T,N011K/G023K/D027S/V154I/G156W/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/N094R/D130A/L264R,P029E/N033D/K074S/L075D/N101D/T189D,Q004D/D027N/E056K/S058M/N233Q/P256T,Q004D/D027S/E056K/S058M/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q,Q004D/N011K/D027S/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,A018K/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/E045F/A049V/L075Q/G156W/V187T,A018K/G023Q/E045F/L075Q/G156W/V187T/T189D,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T,A018K/G023Q/K024A/L075R/N094R/G156W/V187N,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/D130A/G156W/V187N/T189Q/L264R,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027S/E056K/L075R/D111A/G156W/V187N/L264R,D027S/L075R/V154I/G156W/V187N/T189Q/L264R,D027S/P029E/N033D/E045F/N073R/L075D/T189D,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027S/L075Q/N094R/V154I/G156W/T189Q,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/D111A/V154I/G156W/T189Q/L264R,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/V187N/L264R,G023Q/D027S/L075Q/V154I/G156W/V187N/L264R,G023Q/E045F/A049V/V077I/G156W/V187T/T189D,G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,G023Q/F051T/E056K/L075R/G091Q/V187H/I252Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/D027N/E056K/S058M/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/I090F/N233Q/P256T,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,Q004D/N011K/D027Q/S058M/I090F/N233Q/P256T,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,G023Q/E056K/L075Q/D111A/V154I/V187N/T189Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023K/L075Q/D111A/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023Q/D027S/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/D027S/E056K/L075R/D111A/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 7-11 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index <1 in the FabricAdhesion assay compared to Reference sequence SEQ ID NO: 2 are shownbelow. A018K/E045F, A018K/L075D, D027E/G163P, D027S/P256T, E045F/N073R,F051T/L075R, K024A/V154I, L227M/L264R, N033D/E045F, A018K/E045F/N073R,A018K/E045F/V187T, A018K/V154I/G156W, D027N/S058M/P256T,D027Q/S058M/P256T, D027S/L075Q/G091Q, D027S/S058M/P256T,D130A/V154I/G156W, E056K/D130A/L264R, G023Q/K024A/G156W,I090F/N233Q/P256T, N011K/N233Q/P256T, N094R/D130A/V187T,P029E/N033D/E045F, Q004D/D027S/P256T, Q004D/S058M/N233Q,A018K/D111A/G156W/T189Q, A018K/E045F/A049V/G156W,A018K/E045F/L075D/T189D, A018K/P029E/N073R/L075D,D027E/D137Q/L227M/L264R, D027N/S058M/N233Q/P256T,D027Q/E056K/N233Q/P256T, D027S/E045F/L075D/T189D,D027S/E045F/N073R/T189D, D027S/N033D/E045F/N073R,D027S/N033D/L075D/T189D, D027S/P029E/E045F/N073R,D027S/P029E/E045F/T189D, D027S/P029E/N033D/E045F,D048Q/D137Q/G163P/L227M, E045F/A049V/G156W/V187T,E045F/L075Q/G156W/V187T, G023K/E056K/D130A/V187N,G023K/E056K/V187T/L264R, G023Q/K024A/L075R/G156W,L075G/G091Q/V187N/L264R, N011K/D027Q/S058M/P256T,N011K/D027S/N233Q/P256T, N033D/E045F/N073R/T189A,Q004D/D027N/E056K/P256T, Q004D/D027N/S058M/P256T,Q004D/D027Q/N233Q/P256T, Q004D/D027Q/S058M/P256T,Q004D/N011K/D027S/P256T, A018K/D027S/E045F/L075D/T189D,A018K/E045F/N073R/L075D/T189D, A018K/K024A/L075R/D130A/V154I,D027E/D130A/D137Q/G163P/L264R, D027Q/F051T/L075Q/D130A/L264R,D027S/E056K/D111A/V187N/L264R, D048Q/D137Q/G163P/N233Q/L264R,D048Q/S058M/G163P/L227M/L264R, E045F/A049V/L075Q/V187T/T189D,E056K/L075R/D130A/V187N/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/L075R/V187N/L264R,G023K/K024A/D111A/D130A/V187T, G023K/L075Q/V187H/I252Q/L264R,G023Q/D027S/L075G/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,N011K/D027N/S058M/N233Q/P256T, N011K/D027S/I090F/N233Q/P256T,Q004D/N011K/D027N/N233Q/P256T, S058M/D137Q/G163P/L227M/N233Q,V077I/D130A/V154I/G156W/V187N, A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023Q/K024A/L075R/N094R/G156W,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027Q/E056K/S058M/D130A/I252Q/L264R,D027S/E056K/S0581M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023Q/D027S/N094R/V154I/G156W/T189Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/V077I/D130A/V154I/G156W,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027N/S058M/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,G023Q/F051T/E056K/L075R/G091Q/V187H/I252Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,N011K/G023Q/L075Q/V154I/V187N/T189Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

TABLE 7-12 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≤50% of the maximumPI value in the Fabric Adhesion assay compared to Reference sequence SEQID NO: 2 are shown below. A018K/E045F, A018K/L075D, D027E/G163P,E045F/N073R, F051T/L075R, K024A/V154I, L227M/L264R, N033D/E045F,A018K/E045F/N073R, A018K/V154I/G156W, D027S/L075Q/G091Q,D027S/S058M/P256T, E056K/D130A/L264R, I090F/N233Q/P256T,N011K/N233Q/P256T, Q004D/S058M/N233Q, A018K/D111A/G156W/T189Q,A018K/E045F/L075D/T189D, D027E/D137Q/L227M/L264R,D027Q/E056K/N233Q/P256T, D027S/E045F/L075D/T189D,D027S/E045F/N073R/T189D, D027S/N033D/E045F/N073R,D027S/P029E/E045F/N073R, D027S/P029E/N033D/E045F,D048Q/D137Q/G163P/L227M, G023K/E056K/D130A/V187N,G023K/E056K/V187T/L264R, G023Q/K024A/L075R/G156W,L075G/G091Q/V187N/L264R, N033D/E045F/N073R/T189A,Q004D/D027N/S058M/P256T, Q004D/D027Q/N233Q/P256T,Q004D/D027Q/S058M/P256T, Q004D/N011K/D027S/P256T,A018K/D027S/E045F/L075D/T189D, A018K/E045F/N073R/L075D/T189D,A018K/K024A/L075R/D130A/V154I, D027Q/F051T/L075Q/D130A/L264R,D048Q/D137Q/G163P/N233Q/L264R, E045F/A049V/L075Q/V187T/T189D,E056K/L075R/D130A/V187N/L264R, G023K/E056K/L075R/V187N/L264R,G023K/K024A/D111A/D130A/V187T, G023K/L075Q/V187H/I252Q/L264R,G023Q/D027S/L075G/I252Q/L264R, N011K/D027N/S058M/N233Q/P256T,N011K/D027S/I090F/N233Q/P256T, S058M/D137Q/G163P/L227M/N233Q,A018K/D027S/N033D/E045F/N073R/L075D,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/L075G/D130A/V187T/I252Q/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/K024A/V077I/D130A/V154I/G156W,N011K/D027S/E056K/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,G023Q/F051T/E056K/L075R/G091Q/V187H/I252Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,N011K/D027N/E056K/S058M/I090F/N233Q/P256T,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D

Example 8 Cleaning Performance of TLL Combinatorial Variants

The cleaning performance of TLL variants created as described in Example2 was assayed as described in Example 1 (CS-61 microswatch Assay) usingTide® half dose+adjuvant or Tide® full dose. The performance index wascalculated for the variants compared to TLL, SEQ ID NO:1 or thereference sequence SEQ ID NO: 2.

TABLE 8-1 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 in the CS-61 microswatch Assay using Tide ® half dose + adjuvantare shown below. A018K/L075Q, A018K/T189D, D027S/N033D, D130A/L264R,D130A/V187T, G023K/D130A, K024A/L075Q, L075Q/D130A, L075Q/G156W,L075R/L264R, N033D/E045F, P029E/N033D, V187T/L264R, A018K/A049V/L075Q,A018K/D027S/E045F, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/T189D, A018K/E045F/V187T, A018K/G023K/L075Q,A018K/G023Q/L075Q, A018K/L075D/T189D, A018K/L075Q/G156W,A018K/L075Q/V187T, A018K/N033D/L075D, A018K/N073R/L075D,A018K/P029E/T189D, A049V/V187T/T189D, D027E/S058M/G163P,D027S/L075Q/G091Q, D027S/N033D/T189D, D027S/N233Q/P256T,D111A/D130A/L264R, E045F/N073R/L075D, E056K/D130A/T189Q,F051T/I252Q/L264R, G023K/E056K/V187T, G023K/V187N/L264R,G023Q/A049V/T189D, G023Q/D111A/L264R, G023Q/E056K/L075R,G023Q/L075Q/V077I, G023Q/L075Q/V187T, G163P/L227M/L264R,K024A/L075Q/V077I, L075G/D130A/V187H, L075Q/D111A/D130A,L075Q/D130A/V187T, L075Q/G156W/T189D, L075Q/G156W/V187N,L075Q/V077I/T189D, L075Q/V154I/V187T, L075Q/V187N/L264R,L075Q/V187T/L264R, L075Q/V187T/T189D, L075R/D130A/L264R,L075R/D130A/V187T, L075R/V187N/L264R, N033D/E045F/L075D,N033D/E045F/N073R, N033D/N073R/T189D, N073R/L075D/T189D,P029E/E045F/L075D, P029E/L075D/T189D, P029E/N033D/E045F,P029E/N033D/L075D, P029E/N073R/L075D, Q004D/N233Q/P256T,S058M/L227M/L264R, V077I/D130A/V154I, V077I/V187A/T189D,V187N/T189Q/L264R, A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/T189D, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/D130A/G156W/V187T,A018K/E045F/L075Q/T189D, A018K/E045F/L075Q/V187T,A018K/G023Q/A049V/T189D, A018K/G023Q/E045F/T189D,A018K/G023Q/E045F/V187T, A018K/G023Q/L075Q/T189D,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V187T/T189D,A018K/L075Q/G156W/T189D, A018K/L075Q/N094R/D111A,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/N033D/L075D/T189D, A018K/N073R/L075D/T189D,A018K/P029E/N033D/T189D, A049V/L075Q/V077I/T189D,A049V/L075Q/V187T/T189D, D027E/D130A/N233Q/L264R,D027E/D137Q/G163P/L227M, D027E/D137Q/L227M/L264R,D027E/L227M/N233Q/L264R, D027E/S058M/G163P/L264R,D027S/D130A/I252Q/L264R, D027S/E056K/D111A/V154I,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/L075D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D027S/P029E/N033D/T189D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,D111A/D130A/V154I/L264R, D130A/G163P/L227M/L264R,D130A/G163P/N233Q/L264R, E056K/D130A/V187N/L264R,E056K/L075Q/V187N/L264R, F051T/D130A/I252Q/L264R,F051T/L075G/I252Q/L264R, F051T/L075Q/I252Q/L264R,G023K/D027Q/F051T/L075Q, G023K/D130A/G156W/T189Q,G023K/D130A/V187T/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/V187T/L264R, G023K/K024A/V154I/V187N,G023K/L075Q/D130A/V187N, G023K/L075Q/G156W/L264R,G023K/L075Q/G156W/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187N/L264R, G023Q/A049V/L075Q/T189D,G023Q/A049V/L075Q/V077I, G023Q/A049V/V077I/G156W,G023Q/D027S/D111A/G156W, G023Q/E045F/A049V/T189D,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/K024A/L075R/V154I, 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Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/K024A/L075R/D111A/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,A018K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187T/L264R,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/E056K/L075Q/D111A/V154I/V187N/T189Q/L264R,G023Q/E056K/L075Q/N094R/D111A/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 8-2 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 in the CS-61 microswatch Assay using Tide ® halfdose + adjuvant compared to TLL SEQ ID NO: 1 are shown below.D027S/N033D, D130A/L264R, D130A/V187T, L075R/L264R, N033D/E045F,P029E/N033D, V187T/L264R, A018K/E045F/V187T, A018K/L075D/T189D,A018K/P029E/T189D, D027E/S058M/G163P, D027S/L075Q/G091Q,D027S/N033D/T189D, D111A/D130A/L264R, F051T/I252Q/L264R,G023K/E056K/V187T, G023K/V187N/L264R, G023Q/A049V/T189D,G163P/L227M/L264R, L075G/D130A/V187H, L075Q/V187N/L264R,L075Q/V187T/L264R, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187N/L264R, P029E/N033D/E045F, V187N/T189Q/L264R,A018K/D027S/P029E/T189D, A018K/D130A/G156W/V187T,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,D027E/D130A/N233Q/L264R, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/L227M/N233Q/L264R,D027E/S058M/G163P/L264R, D027S/D130A/I252Q/L264R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,D130A/G163P/L227M/L264R, E056K/D130A/V187N/L264R,F051T/D130A/I252Q/L264R, F051T/L075G/I252Q/L264R,F051T/L075Q/I252Q/L264R, G023K/D027Q/F051T/L075Q,G023K/D130A/G156W/T189Q, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/E056K/V187T/L264R,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023Q/A049V/V077I/G156W, G023Q/L075Q/D130A/L264R,G023Q/L075Q/V187T/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075G/V187H/I252Q/L264R,L075R/D130A/V187T/L264R, N011K/D027S/N233Q/P256T,P029E/N073R/L075D/T189D, S058M/D137Q/G163P/N233Q,S058M/G163P/L227M/L264R, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, A018K/D027S/P029E/N033D/L075D,A018K/D130A/G156W/V187N/L264R, A018K/E045F/N073R/L075D/T189D,A018K/G023K/D130A/V154I/G156W, A018K/G023K/K024A/D130A/G156W,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/V077I/D130A/G156W, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D027E/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027E/S058M/G163P/L227M/L264R,D027S/E056K/D111A/V187N/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075G/G091Q/I252Q/L264R, D027S/L075R/V154I/T189Q/L264R,D048Q/D130A/G163P/L227M/L264R, D048Q/D137Q/G163P/L227M/L264R,D048Q/D137Q/G163P/N233Q/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075G/V187N/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/L075Q/V187N/I252Q/L264R, F051T/S058M/L075Q/G091Q/I252Q,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187T/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/E056K/V187N/I252Q/L264R,G023Q/F051T/L075Q/G091Q/I252Q, G023Q/L075G/G091Q/I252Q/L264R,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, L075G/D130A/V187T/I252Q/L264R,N011K/G023K/D111A/G156W/L264R, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, S058M/D137Q/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/V077I/V154I/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/F051T/L075Q/D130A/I252Q/L264R,D027S/L075Q/G091Q/V187H/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D137Q/G163P/L227M/L264R,D048Q/S058M/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/G091Q/V187N/I252Q/L264R,G023Q/F051T/L075G/G091Q/V187N/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/G091Q/D130A/V187H/I252Q/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/S058M/L075Q/G091Q/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/D048Q/S058M/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 8-3 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value in the CS-61 microswatchAssay using Tide ® half dose + adjuvant compared to TLL SEQ ID NO: 1 areshown below. P029E/N033D, A018K/L075D/T189D, A018K/P029E/T189D,D027S/N033D/T189D, G023K/V187N/L264R, G023Q/A049V/T189D,L075G/D130A/V187H, L075Q/V187N/L264R, L075R/D130A/V187T,L075R/V187N/L264R, P029E/N033D/E045F, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,D027E/D137Q/G163P/L227M, D027E/L227M/N233Q/L264R,D027S/N033D/L075D/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, G023K/D027Q/F051T/L075Q,G023K/D130A/V187T/L264R, G023Q/A049V/V077I/G156W,L075G/D130A/V187T/I252Q, L075R/D130A/V187T/L264R,P029E/N073R/L075D/T189D, S058M/D137Q/G163P/N233Q,A018K/G023K/K024A/D130A/G156W, A018K/G023Q/K024A/D130A/V187Q,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027E/D130A/G163P/N233Q/L264R, D027E/D137Q/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187T/L264R,K024A/L075R/V154I/G156W/V187Q, N011K/G023K/D111A/G156W/L264R,S058M/D130A/G163P/L227M/L264R, A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/V077I/D130A/G156W/V187N,K024A/L075Q/V077I/N094R/D130A/V187N,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 8-4 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 in the CS-61 microswatch Assay using Tide ® halfdose + adjuvant are shown below. A018K/L075Q, A018K/T189D, G023K/D130A,K024A/L075Q, L075Q/D130A, L075Q/G156W, P029E/N033D, A018K/A049V/L075Q,A018K/D027S/N073R, A018K/D027S/T189D, A018K/E045F/T189D,A018K/G023K/L075Q, A018K/G023Q/L075Q, A018K/L075Q/G156W,A018K/L075Q/V187T, A018K/N033D/L075D, A018K/N073R/L075D,A018K/P029E/T189D, A049V/V187T/T189D, D027S/N033D/T189D,D027S/N233Q/P256T, E056K/D130A/T189Q, G023K/V187N/L264R,G023Q/A049V/T189D, G023Q/D111A/L264R, G023Q/E056K/L075R,G023Q/L075Q/V077I, G023Q/L075Q/V187T, K024A/L075Q/V077I,L075G/D130A/V187H, L075Q/D130A/V187T, L075Q/G156W/T189D,L075Q/G156W/V187N, L075Q/V077I/T189D, L075Q/V154I/V187T,L075Q/V187T/T189D, L075R/D130A/V187T, L075R/V187N/L264R,N073R/L075D/T189D, P029E/E045F/L075D, P029E/L075D/T189D,P029E/N033D/E045F, P029E/N073R/L075D, Q004D/N233Q/P256T,S058M/L227M/L264R, V077I/V187A/T189D, A018K/A049V/L075Q/T189D,A018K/A049V/L075Q/V187T, A018K/D027S/N033D/L075D,A018K/D027S/P029E/T189D, A018K/E045F/L075Q/T189D,A018K/G023Q/E045F/T189D, A018K/G023Q/E045F/V187T,A018K/G023Q/L075Q/T189D, A018K/G023Q/L075Q/V077I,A018K/G023Q/L075Q/V187T, A018K/G023Q/V187T/T189D,A018K/L075Q/N094R/D111A, A018K/L075Q/N094R/V187Q,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A049V/L075Q/V077I/T189D,A049V/L075Q/V187T/T189D, D027E/D137Q/G163P/L227M,D027S/N033D/L075D/T189D, D027S/P029E/E045F/L075D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D027S/P029E/N033D/T189D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, D130A/G163P/N233Q/L264R,E056K/L075Q/V187N/L264R, G023K/D130A/V187T/L264R,G023K/L075Q/D130A/V187N, G023K/L075Q/G156W/L264R,G023K/L075Q/G156W/V187N, G023K/L075R/V187N/L264R,G023Q/A049V/L075Q/T189D, G023Q/A049V/L075Q/V077I,G023Q/D027S/D111A/G156W, G023Q/E045F/A049V/T189D,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/K024A/L075R/V154I, G023Q/L075Q/G156W/V187N,G023Q/L075Q/G156W/V187T, K024A/L075Q/V187T/T189Q,K024A/L075R/G156W/V187N, L075G/D130A/V187T/I252Q,L075Q/D130A/G156W/V187N, L075Q/V077I/D130A/V187Q,L075Q/V077I/G156W/V187N, L075Q/V077I/N094R/G156W,L075Q/V077I/V154I/V187Q, L075Q/V077I/V187T/T189D,L075R/D130A/V154I/L264R, N011K/E056K/S058M/P256T,P029E/E045F/L075D/T189D, P029E/E045F/N073R/L075D,P029E/E045F/N073R/T189D, P029E/N033D/E045F/L075D,P029E/N033D/L075D/T189D, P029E/N073R/L075D/T189D,Q004D/D027S/I090F/P256T, Q004D/N011K/D027N/P256T,A018K/D027S/E045F/N073R/D137V, A018K/D027S/N033D/E045F/T189D,A018K/D027S/N033D/L075D/T189D, A018K/D027S/N073R/L075D/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N073R/L075D,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/L075R/N094R,A018K/G023K/L075Q/D130A/V154I, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/L075Q/G156W/V187T, A018K/G023Q/L075Q/V187T/T189D,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075Q/V154I/G156W,A018K/K024A/L075R/N094R/D130A, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/G156W/V187N,A018K/L075Q/V077I/N094R/G156W, A018K/N033D/E045F/L075D/T189D,A018K/P029E/E045F/N073R/L075D, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/L264R, D027E/D137Q/G163P/N233Q/L264R,D027S/N033D/N073R/L075D/T189D, D027S/P029E/N033D/N073R/L075D,D027S/P029E/N073R/L075D/T189D, D048Q/D130A/D137Q/G163P/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/D130A/V187H/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/D027S/E056K/V187T/L264R, G023K/D111A/V154I/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/D130A/G156W/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187T/L264R, G023Q/A049V/L075Q/G156W/T189D,G023Q/A049V/L075Q/G156W/V187T, G023Q/A049V/L075Q/V187T/T189D,G023Q/D027S/V154I/V187N/L264R, G023Q/E045F/L075Q/V077I/V187T,G023Q/K024A/L075Q/G156W/V187N, G023Q/K024A/L075Q/V077I/V187Q,G023Q/K024A/L075R/G156W/V187Q, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/V077I/G156W/V187N, G091Q/D130A/V187H/I252Q/L264R,K024A/L075Q/D111A/G156W/V187T, K024A/L075Q/V077I/G156W/V187N,K024A/L075Q/V077I/V154I/V187N, K024A/L075R/V154I/G156W/V187Q,L075Q/D111A/V187N/T189Q/L264R, L075Q/V154I/V187N/T189Q/L264R,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, L075R/G156W/V187T/T189Q/L264R,N011K/G023K/D111A/G156W/L264R, N011K/G023K/E056K/L075Q/T189Q,P029E/N033D/E045F/L075D/T189D, P029E/N033D/K074S/L075D/T189D,P029E/N033D/N073R/L075D/T189D, Q004D/D027Q/E056K/N233Q/P256T,Q004D/N011K/D027Q/E056K/N233Q, Q004D/N011K/I090F/N233Q/P256T,A018K/A049V/L075Q/G156W/V187T/T189D,A018K/D027S/N033D/E045F/L075D/T189D,A018K/D027S/P029E/N073R/L075D/T189D,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075Q/G156W/V187N/L264R,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/A049V/L075Q/V077I/V187T,A018K/G023Q/E045F/A049V/L075Q/T189D,A018K/G023Q/L075Q/G156W/V187T/T189D,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/L075Q/V077I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/N094R/V154I/V187N,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/L075R/D130A/V154I/G156W/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D027S/P029E/N033D/E045F/N073R/T189D,D027S/P029E/N033D/N073R/L075D/T189D,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/N094R/V154I/V187T/L264R,G023K/L075Q/V077I/D130A/G156W/V187Q,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/A049V/L075Q/G156W/V187T/T189D,G023Q/D027Q/E056K/L075R/I252Q/L264R,G023Q/D027S/F051T/L075Q/V187T/I252Q,G023Q/E045F/L075Q/V077I/G156W/V187T,G023Q/E045F/L075Q/V077I/V187T/T189D,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,K024A/L075Q/V154I/V187N/T189Q/L264R,K024A/N094R/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/V154I/V187N/T189Q,L075Q/N094R/D111A/G156W/T189Q/L264R,N011K/D027S/S058M/I090F/N233Q/P256T,N011K/E056K/L075Q/D130A/V187N/T189Q,N011K/G023K/D027S/V154I/G156W/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,P029E/N033D/K074S/L075D/N101D/T189D,Q004D/N011K/D027Q/E056K/S058M/N233Q,A018K/G023K/K024A/L075Q/V077I/N094R/G156W,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/K024A/L075R/V154I/G156W/V187Q,A018K/G023K/K024A/N094R/V154I/V187T/L264R,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075Q/V077I/N094R/V154I,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/D111A/G156W/V187N/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/D111A/V154I/V187T/T189Q/L264R,A018K/L075Q/N094R/D111A/V154I/V187T/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027S/F051T/L075G/G091Q/V187T/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/L075Q/N094R/V154I/G156W/T189Q,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/K024A/L075Q/D111A/V154I/G156W/V187N,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023K/L075Q/N094R/V154I/G156W/V187T/L264R,G023K/L075R/N094R/V154I/G156W/V187N/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/E045F/A049V/V077I/G156W/V187T/T189D,G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/L075R/G091Q/V187N/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,Q004D/N011K/D027Q/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/K024A/L075R/D111A/V154I/V187T/L264R,A018K/G023K/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,A018K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187T/L264R,G023K/K024A/L075R/N094R/V154I/G156W/V187T/T189Q,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/E056K/S058M/L075R/G091Q/V187N/I252Q,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/E056K/L075Q/D111A/V154I/V187N/T189Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/D027S/L075Q/N094R/V154I/G156W/T189Q,N011K/G023Q/D027S/N094R/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/N094R/V154I/G156W/T189Q/L264R,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/L075Q/N094R/D111A/V154I/G156W/T189Q/L264R,A018K/G023Q/D027S/P029E/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/G156W/V187H,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/P029E/S058M/V077I/R108K/H135F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 8-5 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 in the CS-61microswatch Assay using Tide ® half dose + adjuvant compared toReference sequence SEQ ID NO: 2 are shown below. P029E/N033D,A018K/P029E/T189D, D027S/N033D/T189D, G023K/V187N/L264R,G023Q/A049V/T189D, L075G/D130A/V187H, L075R/D130A/V187T,L075R/V187N/L264R, P029E/N033D/E045F, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,D027E/D137Q/G163P/L227M, D027S/N033D/L075D/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D130A/V187T/L264R, L075G/D130A/V187T/I252Q,P029E/N073R/L075D/T189D, A018K/G023K/K024A/D130A/G156W,A018K/K024A/N094R/D130A/V187N, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E056K/L075R/D130A/V187T/L264R, G023K/D027S/E056K/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187T/L264R, K024A/L075R/V154I/G156W/V187Q,N011K/G023K/D111A/G156W/L264R, A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/N094R/D111A/G156W/V187T/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 8-6 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value in the CS-61 microswatch Assay using Tide ® half dose +adjuvant compared to Reference sequence SEQ ID NO: 2 are shown below.D027S/N033D/T189D, G023K/V187N/L264R, L075R/D130A/V187T,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,P029E/N073R/L075D/T189D, A018K/K024A/N094R/D130A/V187N,D027E/D130A/G163P/N233Q/L264R, D027E/D137Q/G163P/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/L075R/D130A/V187T/L264R,K024A/L075R/V154I/G156W/V187Q, A018K/K024A/L075Q/N094R/V154I/G156W,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/N094R/D111A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 8-7 TLL variants with Performance Index >1 compared to TLL SEQ IDNO: 1 in the CS-61 microswatch Assay using Tide ® full dose are shownbelow. A018K/D027S, A018K/L075Q, A018K/T189D, D027N/E056K, D027S/N033D,D130A/L264R, D130A/V187N, E045F/T189D, G023K/D130A, G023K/L264R,K024A/L075Q, K024A/V154I, L075Q/D130A, L075Q/G156W, L227M/L264R,N033D/E045F, P029E/N033D, V187T/L264R, A018K/A049V/L075Q,A018K/D027S/E045F, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/T189D, A018K/G023K/G156W, A018K/G023K/L075Q,A018K/G023Q/L075Q, A018K/K024A/V154I, A018K/L075D/T189D,A018K/L075Q/G156W, A018K/L075Q/V187T, A018K/N033D/L075D,A018K/N073R/L075D, A018K/P029E/T189D, A018K/V154I/G156W,A049V/V187T/T189D, D027E/S058M/G163P, D027Q/N233Q/P256T,D027S/N033D/T189D, D027S/N233Q/P256T, D111A/D130A/L264R,E045F/N073R/L075D, E056K/D130A/T189Q, G023K/D130A/L264R,G023K/D130A/V187T, G023K/E056K/V187T, G023K/V187N/L264R,G023Q/A049V/T189D, G023Q/D111A/L264R, G023Q/E056K/L075R,G023Q/K024A/G156W, G023Q/L075Q/V077I, G023Q/L075Q/V187T,K024A/L075Q/G156W, K024A/L075Q/V077I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/D130A/V187T, L075Q/G156W/T189D,L075Q/G156W/V187N, L075Q/G156W/V187T, L075Q/V077I/T189D,L075Q/V154I/V187T, L075Q/V187N/L264R, L075Q/V187T/L264R,L075Q/V187T/T189D, L075R/D130A/L264R, L075R/D130A/V187T,L075R/V187N/L264R, L075R/V187T/L264R, N011K/D027S/S058M,N033D/E045F/N073R, N033D/N073R/T189D, N073R/L075D/T189D,P029E/E045F/L075D, P029E/L075D/T189D, P029E/N033D/E045F,P029E/N033D/L075D, P029E/N073R/L075D, S058M/L227M/L264R,V077I/D130A/V154I, V077I/V187A/T189D, V187N/T189Q/L264R,A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/N073R, A018K/D027S/E045F/T189D,A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/E045F/L075D/T189D, A018K/E045F/L075Q/T189D,A018K/E045F/L075Q/V187T, A018K/G023K/D111A/T189Q,A018K/G023Q/A049V/T189D, A018K/G023Q/A049V/V187T,A018K/G023Q/E045F/T189D, A018K/G023Q/E045F/V187T,A018K/G023Q/G156W/V187T, A018K/G023Q/L075Q/T189D,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/L075Q/G156W/T189D,A018K/L075Q/G156W/V187T, A018K/L075Q/N094R/D111A,A018K/L075Q/N094R/V187Q, A018K/L075Q/V077I/N094R,A018K/L075Q/V187T/T189D, A018K/N033D/L075D/T189D,A018K/N073R/L075D/T189D, A018K/P029E/N033D/T189D,A018K/P029E/N073R/L075D, A049V/L075Q/V077I/T189D,A049V/L075Q/V187T/T189D, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/L227M/N233Q/L264R,D027S/E056K/D111A/V154I, D027S/N033D/E045F/T189D,D027S/N033D/L075D/T189D, D027S/N033D/N073R/T189D,D027S/P029E/E045F/L075D, D027S/P029E/E045F/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D027S/P029E/N033D/T189D,D048Q/D130A/G163P/L264R, D111A/D130A/V154I/L264R,D130A/G163P/L227M/L264R, D130A/G163P/N233Q/L264R,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,E056K/L075Q/V187N/L264R, G023K/D027Q/F051T/L075Q,G023K/D027S/L075Q/L264R, G023K/D130A/V187T/L264R,G023K/E056K/D130A/L264R, G023K/E056K/V187T/L264R,G023K/K024A/V154I/V187N, G023K/L075Q/D130A/V187N,G023K/L075Q/G156W/L264R, G023K/L075Q/G156W/V187N,G023K/L075R/D130A/L264R, G023K/L075R/V187N/L264R,G023K/L075R/V187T/L264R, G023Q/A049V/L075Q/T189D,G023Q/A049V/L075Q/V077I, G023Q/D027S/D111A/G156W,G023Q/E045F/A049V/T189D, G023Q/E045F/A049V/V187T,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/K024A/L075R/G156W, G023Q/K024A/L075R/V154I,G023Q/L075Q/G156W/V187N, G023Q/L075Q/G156W/V187T,G023Q/L075Q/V187T/L264R, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, K024A/L075Q/V187T/T189Q,K024A/L075R/G156W/V187N, L075G/D130A/V187T/I252Q,L075Q/D130A/G156W/V187N, L075Q/D130A/V154I/G156W,L075Q/V077I/D130A/V187Q, L075Q/V077I/G156W/V187N,L075Q/V077I/N094R/G156W, L075Q/V077I/V154I/V187Q,L075Q/V077I/V187T/T189D, L075R/D111A/V154I/G156W,L075R/D130A/V154I/L264R, N011K/D027N/S058M/P256T,N011K/D027S/E056K/P256T, N011K/D027S/N233Q/P256T,N011K/G023K/L075Q/D111A, P029E/E045F/L075D/T189D,P029E/E045F/N073R/L075D, P029E/E045F/N073R/T189D,P029E/N033D/E045F/L075D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, Q004D/D027S/I090F/P256T,Q004D/N011K/D027N/P256T, Q004D/N011K/D027Q/N233Q,S058M/D137Q/G163P/N233Q, S058M/G163P/N233Q/L264R,S058M/L075Q/G091Q/I252Q, A018K/D027S/E045F/N073R/D137V,A018K/D027S/N033D/E045F/T189D, A018K/D027S/N033D/L075D/T189D,A018K/D027S/N073R/L075D/T189D, A018K/D027S/P029E/E045F/T189D,A018K/D027S/P029E/N033D/L075D, A018K/D027S/P029E/N073R/L075D,A018K/D130A/G156W/V187N/L264R, A018K/E045F/A049V/L075Q/V187T,A018K/E045F/N073R/L075D/T189D, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/K024A/L075R/N094R,A018K/G023K/L075Q/D130A/V154I, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/A049V/L075Q/G156W, A018K/G023Q/A049V/L075Q/V077I,A018K/G023Q/A049V/L075Q/V187T, A018K/G023Q/A049V/V077I/V187T,A018K/G023Q/E045F/A049V/L075Q, A018K/G023Q/E045F/A049V/V187T,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/E045F/V077I/T189D,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/D130A/V187Q,A018K/G023Q/K024A/L075Q/G156W, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/D130A/G156W,A018K/G023Q/V077I/V187T/T189D, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075Q/V154I/G156W, A018K/K024A/L075Q/V154I/T189D,A018K/K024A/L075R/D130A/V154I, A018K/K024A/L075R/N094R/D130A,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, A018K/L075Q/N094R/G156W/V187N,A018K/L075Q/V077I/N094R/G156W, A018K/N033D/E045F/L075D/T189D,A018K/P029E/E045F/N073R/L075D, A018K/P029E/N033D/N073R/L075D,A018K/V077I/G156W/V187T/T189D, D027E/D048Q/S058M/G163P/N233Q,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/L264R, D027E/D137Q/G163P/N233Q/L264R,D027E/S058M/D137Q/G163P/N233Q, D027N/E056K/S058M/N233Q/P256T,D027N/S058M/I090F/N233Q/P256T, D027S/E056K/D111A/V187N/L264R,D027S/E056K/S058M/N233Q/P256T, D027S/L075G/D130A/I252Q/L264R,D027S/L075Q/D111A/D130A/V187N, D027S/L075R/V154I/T189Q/L264R,D027S/N033D/E045F/N073R/L075D, D027S/N033D/N073R/L075D/T189D,D027S/P029E/N033D/E045F/N073R, D027S/P029E/N033D/N073R/L075D,D027S/P029E/N073R/L075D/T189D, D048Q/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/N233Q/L264R, D048Q/S058M/D137Q/G163P/L264R,D048Q/S058M/D137Q/G163P/N233E, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/D130A/V187H/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, E056K/L075R/D130A/V187T/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/D111A/V154I/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/D130A/V154I/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187T/L264R,G023K/N094R/G156W/V187N/T189Q, G023Q/A049V/L075Q/G156W/T189D,G023Q/A049V/L075Q/G156W/V187T, G023Q/A049V/L075Q/V187T/T189D,G023Q/D027S/V154I/V187N/L264R, G023Q/E045F/A049V/G156W/T189D,G023Q/E045F/L075Q/G156W/T189D, G023Q/E045F/L075Q/V077I/V187T,G023Q/K024A/L075Q/D130A/G156W, G023Q/K024A/L075Q/G156W/V187N,G023Q/K024A/L075Q/G156W/V187Q, G023Q/K024A/L075Q/V077I/V187Q,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/G156W/V187Q,G023Q/K024A/L075R/V077I/D130A, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/V077I/D130A/G156W, G023Q/L075Q/V077I/G156W/V187N,G091Q/D130A/V187H/I252Q/L264R, K024A/L075Q/D111A/G156W/V187T,K024A/L075Q/V077I/G156W/V187N, K024A/L075Q/V077I/V154I/V187N,K024A/L075R/V154I/G156W/V187Q, L075Q/D111A/D130A/V187T/T189Q,L075Q/D111A/V187N/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075Q/V154I/V187N/T189Q/L264R,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, L075R/G156W/V187T/T189Q/L264R,L075R/V077I/N094R/V154I/G156W, N011K/D027N/E056K/N233Q/P256T,N011K/D027N/E056K/S058M/P256T, N011K/D027Q/E056K/S058M/N233Q,N011K/G023K/D111A/G156W/L264R, N011K/G023K/E056K/L075Q/T189Q,P029E/N033D/E045F/L075D/T189D, P029E/N033D/K074S/L075D/T189D,P029E/N033D/N073R/L075D/T189D, Q004D/D027S/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/N233Q, S058M/D130A/D137Q/G163P/L264R,S058M/G163P/L227M/N233Q/L264R, 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F/G156W,A018K/G023Q/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/F051T/E056K/S058M/L075Q/G091Q/V187N/L264R,G023K/D027S/F051T/E056K/S058M/L075R/G091Q/V187H/I252Q,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/F051T/E056K/L075G/G091Q/V187N/I252Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023Q/L075Q/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/E045F/A049V/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/E045F/A049V/S058M/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023Q/D027S/S058M/L075Q/V077I/R108K/H135F/G156W/V187H,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023K/L075R/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T,N011K/G023Q/D027S/L075Q/N094R/D111A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/V187T/T189D,A018K/G023Q/D027S/P029E/E045F/A049V/S058M/N073S/L075Q/R108K/H135F/V187T/T189D,A018K/G023Q/D027S/P029E/N033D/E045F/A049V/S058M/N073S/K074S/L075Q/V077I/N101D/R108K/H135F/D137V/G156W/V187T/T189D

TABLE 8-8 TLL variants with Performance Index for expression ≥0.5 andPerformance Index >1 in the CS-61 microswatch Assay using Tide ® fulldose compared to TLL SEQ ID NO: 1 are shown below. D027S/N033D,D130A/L264R, D130A/V187N, G023K/L264R, K024A/V154I, L227M/L264R,N033D/E045F, P029E/N033D, V187T/L264R, A018K/K024A/V154I,A018K/L075D/T189D, A018K/P029E/T189D, A018K/V154I/G156W,D027E/S058M/G163P, D027S/N033D/T189D, D111A/D130A/L264R,G023K/D130A/L264R, G023K/D130A/V187T, G023K/E056K/V187T,G023K/V187N/L264R, G023Q/A049V/T189D, G023Q/K024A/G156W,L075G/D130A/V187H, L075Q/V187N/L264R, L075Q/V187T/L264R,L075R/D130A/L264R, L075R/D130A/V187T, L075R/V187N/L264R,L075R/V187T/L264R, P029E/N033D/E045F, V187N/T189Q/L264R,A018K/D027S/P029E/T189D, A018K/E045F/L075D/T189D,A018K/G023K/D111A/T189Q, A018K/G023Q/A049V/V187T,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,A018K/P029E/N073R/L075D, D027E/D137Q/G163P/L227M,D027E/D137Q/L227M/L264R, D027E/L227M/N233Q/L264R,D027S/N033D/E045F/T189D, D027S/N033D/L075D/T189D,D027S/N033D/N073R/T189D, D027S/P029E/E045F/T189D,D027S/P029E/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,D130A/G163P/L227M/L264R, E056K/D130A/V187N/L264R,E056K/D130A/V187T/L264R, G023K/D027Q/F051T/L075Q,G023K/D130A/V187T/L264R, G023K/E056K/V187T/L264R,G023K/K024A/V154I/V187N, G023K/L075R/D130A/L264R,G023K/L075R/V187T/L264R, G023Q/K024A/L075R/G156W,G023Q/L075Q/V187T/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, N011K/D027S/N233Q/P256T,P029E/N073R/L075D/T189D, S058M/D137Q/G163P/N233Q,S058M/G163P/N233Q/L264R, S058M/L075Q/G091Q/I252Q,A018K/D027S/P029E/N033D/L075D, A018K/D130A/G156W/V187N/L264R,A018K/E045F/N073R/L075D/T189D, A018K/G023K/D130A/V154I/G156W,A018K/G023K/K024A/D130A/G156W, A018K/G023K/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/K024A/L075Q/G156W,A018K/G023Q/V077I/D130A/G156W, A018K/K024A/L075R/D130A/V154I,A018K/K024A/N094R/D130A/V187N, A018K/P029E/N033D/N073R/L075D,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D027E/S058M/D137Q/G163P/N233Q,D027S/E056K/D111A/V187N/L264R, D027S/L075G/D130A/I252Q/L264R,D027S/L075R/V154I/T189Q/L264R, D048Q/S058M/D130A/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233E, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/L075R/D130A/V187T/L264R,F051T/L075G/V187N/I252Q/L264R, F051T/L075Q/G091Q/D130A/L264R,F051T/S058M/L075Q/G091Q/I252Q, G023K/D027S/E056K/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187H/I252Q/L264R,G023K/L075R/D130A/V187T/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/K024A/L075Q/G156W/V187Q, G023Q/L075Q/V077I/D130A/G156W,K024A/L075R/V154I/G156W/V187Q, N011K/G023K/D111A/G156W/L264R,S058M/D130A/D137Q/G163P/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/D027S/N033D/N073R/L075D/T189D,A018K/D027S/P029E/N033D/E045F/L075D,A018K/G023K/K024A/V077I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/A049V/G156W/V187T/T189D,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/D111A/V187N/T189Q/L264R,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/G091Q/V187N/I252Q,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/E056K/S058M/V187T/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D137Q/G163P/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027Q/F051T/E056K/S058M/L075Q,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/N094R/D111A/G156W/V187T/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/V077I/D130A/V154I/G156W,K024A/L075Q/V077I/N094R/D130A/V187N,Q004D/N011K/D027N/I090F/N233Q/P256T,Q004D/N011K/D027S/S058M/N233Q/P256T,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075Q/N094R/V154I/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,G023K/D027S/F051T/S058M/G091Q/V187N/I252Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075R/N094R/V154I/G156W/V187N/T189Q,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/G156W/V187T

TABLE 8-9 TLL variants with Performance Index for expression ≥0.5 andPerformance Index ≥50% of the maximum PI value in the CS-61 microswatchAssay using Tide ® full dose compared to TLL SEQ ID NO: 1 are shownbelow. A018K/P029E/T189D, A018K/V154I/G156W, D027S/N033D/T189D,G023K/V187N/L264R, L075R/D130A/V187T, L075R/V187N/L264R,A018K/D027S/P029E/T189D, A018K/L075Q/V077I/N094R,A018K/N033D/L075D/T189D, D027S/P029E/N033D/E045F,D027S/P029E/N033D/L075D, D048Q/D130A/G163P/L264R,G023K/D130A/V187T/L264R, P029E/N073R/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/N094R/D130A/V187N, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/L075R/D130A/V187T/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187T/L264R, K024A/L075R/V154I/G156W/V187Q,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/N094R/D111A/G156W/V187T/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,N011K/A018K/K024A/V077I/V154I/G156W/T189Q,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,N011K/A018K/G023K/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 8-10 TLL variants with Performance Index >1 compared to Referencesequence SEQ ID NO: 2 in the CS-61 microswatch Assay using Tide ® fulldose are shown below. A018K/L075Q, A018K/T189D, E045F/T189D,G023K/D130A, K024A/L075Q, L075Q/D130A, L075Q/G156W, L227M/L264R,A018K/A049V/L075Q, A018K/D027S/N073R, A018K/D027S/T189D,A018K/E045F/T189D, A018K/G023K/L075Q, A018K/G023Q/L075Q,A018K/L075D/T189D, A018K/L075Q/G156W, A018K/L075Q/V187T,A018K/N033D/L075D, A018K/N073R/L075D, A018K/P029E/T189D,A018K/V154I/G156W, D027S/N033D/T189D, D027S/N233Q/P256T,E056K/D130A/T189Q, G023K/V187N/L264R, G023Q/A049V/T189D,G023Q/D111A/L264R, G023Q/E056K/L075R, G023Q/L075Q/V077I,G023Q/L075Q/V187T, K024A/L075Q/G156W, K024A/L075Q/V077I,L075G/D130A/V187H, L075Q/D130A/V187T, L075Q/G156W/T189D,L075Q/G156W/V187N, L075Q/G156W/V187T, L075Q/V077I/T189D,L075Q/V154I/V187T, L075Q/V187N/L264R, L075Q/V187T/T189D,L075R/D130A/V187T, L075R/V187N/L264R, N073R/L075D/T189D,P029E/E045F/L075D, P029E/L075D/T189D, P029E/N033D/E045F,P029E/N033D/L075D, P029E/N073R/L075D, S058M/L227M/L264R,V077I/D130A/V154I, V077I/V187A/T189D, V187N/T189Q/L264R,A018K/A049V/L075Q/T189D, A018K/A049V/L075Q/V187T,A018K/D027S/E045F/N073R, A018K/D027S/E045F/T189D,A018K/D027S/N033D/L075D, A018K/D027S/P029E/T189D,A018K/E045F/L075Q/V187T, A018K/G023Q/A049V/T189D,A018K/G023Q/E045F/T189D, A018K/G023Q/E045F/V187T,A018K/G023Q/G156W/V187T, A018K/G023Q/L075Q/T189D,A018K/G023Q/L075Q/V077I, A018K/G023Q/L075Q/V187T,A018K/G023Q/L075R/D130A, A018K/G023Q/V077I/V187T,A018K/G023Q/V187T/T189D, A018K/L075Q/G156W/T189D,A018K/L075Q/N094R/D111A, A018K/L075Q/N094R/V187Q,A018K/L075Q/V077I/N094R, A018K/L075Q/V187T/T189D,A018K/N033D/L075D/T189D, A018K/N073R/L075D/T189D,A018K/P029E/N033D/T189D, A049V/L075Q/V187T/T189D,D027S/E056K/D111A/V154I, D027S/N033D/L075D/T189D,D027S/P029E/E045F/L075D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D027S/P029E/N033D/T189D, D048Q/D130A/G163P/L264R,D111A/D130A/V154I/L264R, D130A/G163P/L227M/L264R,D130A/G163P/N233Q/L264R, E056K/L075Q/V187N/L264R,G023K/D027S/L075Q/L264R, G023K/D130A/V187T/L264R,G023K/L075Q/D130A/V187N, G023K/L075Q/G156W/L264R,G023K/L075Q/G156W/V187N, G023K/L075R/V187N/L264R,G023Q/A049V/L075Q/T189D, G023Q/A049V/L075Q/V077I,G023Q/D027S/D111A/G156W, G023Q/E045F/A049V/T189D,G023Q/E045F/L075Q/G156W, G023Q/E045F/L075Q/V187T,G023Q/K024A/L075R/V154I, G023Q/L075Q/G156W/V187N,G023Q/L075Q/G156W/V187T, K024A/L075Q/D130A/G156W,K024A/L075Q/V187T/T189Q, K024A/L075R/G156W/V187N,L075Q/D130A/G156W/V187N, L075Q/D130A/V154I/G156W,L075Q/V077I/D130A/V187Q, L075Q/V077I/G156W/V187N,L075Q/V077I/N094R/G156W, L075Q/V077I/V154I/V187Q,L075Q/V077I/V187T/T189D, L075R/D111A/V154I/G156W,L075R/D130A/V154I/L264R, N011K/D027N/S058M/P256T,N011K/D027S/E056K/P256T, N011K/G023K/L075Q/D111A,P029E/E045F/L075D/T189D, P029E/E045F/N073R/L075D,P029E/E045F/N073R/T189D, P029E/N033D/L075D/T189D,P029E/N073R/L075D/T189D, Q004D/D027S/I090F/P256T,Q004D/N011K/D027N/P256T, Q004D/N011K/D027Q/N233Q,A018K/D027S/E045F/N073R/D137V, A018K/D027S/N033D/E045F/T189D,A018K/D027S/N033D/L075D/T189D, A018K/D027S/N073R/L075D/T189D,A018K/D027S/P029E/E045F/T189D, A018K/D027S/P029E/N033D/L075D,A018K/E045F/A049V/L075Q/V187T, A018K/G023K/K024A/L075R/N094R,A018K/G023K/L075Q/D130A/V154I, A018K/G023Q/A049V/L075Q/G156W,A018K/G023Q/A049V/L075Q/V077I, A018K/G023Q/A049V/L075Q/V187T,A018K/G023Q/A049V/V077I/V187T, A018K/G023Q/E045F/A049V/L075Q,A018K/G023Q/E045F/L075Q/V187T, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/L075Q/G156W, A018K/G023Q/L075Q/G156W/V187T,A018K/G023Q/L075Q/V187T/T189D, A018K/G023Q/V077I/V187T/T189D,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075Q/V154I/G156W,A018K/K024A/L075R/D130A/V154I, A018K/K024A/L075R/N094R/D130A,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/G156W/V187N, A018K/L075Q/V077I/N094R/G156W,A018K/N033D/E045F/L075D/T189D, A018K/P029E/E045F/N073R/L075D,A018K/P029E/N033D/N073R/L075D, D027E/D048Q/S058M/G163P/N233Q,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/L227M/L264R, D027E/D137Q/G163P/N233Q/L264R,D027S/L075Q/D111A/D130A/V187N, D027S/L075R/V154I/T189Q/L264R,D027S/N033D/E045F/N073R/L075D, D027S/N033D/N073R/L075D/T189D,D027S/P029E/N033D/E045F/N073R, D027S/P029E/N033D/N073R/L075D,D027S/P029E/N073R/L075D/T189D, D048Q/D130A/D137Q/G163P/L264R,D048Q/S058M/D137Q/G163P/L264R, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/D130A/V187H/I252Q/L264R,E056K/L075Q/G156W/T189Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/D027S/E056K/V187T/L264R, G023K/D111A/V154I/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/D130A/G156W/L264R, G023K/L075Q/D130A/V154I/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075Q/V187H/I252Q/L264R,G023K/L075R/D130A/V187T/L264R, G023K/N094R/G156W/V187N/T189Q,G023Q/A049V/L075Q/G156W/T189D, G023Q/A049V/L075Q/G156W/V187T,G023Q/A049V/L075Q/V187T/T189D, G023Q/D027S/V154I/V187N/L264R,G023Q/E045F/L075Q/G156W/T189D, G023Q/E045F/L075Q/V077I/V187T,G023Q/K024A/L075Q/D130A/G156W, G023Q/K024A/L075Q/G156W/V187N,G023Q/K024A/L075Q/V077I/V187Q, G023Q/K024A/L075R/G156W/V187Q,G023Q/K024A/L075R/V077I/D130A, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/V077I/G156W/V187N, G091Q/D130A/V187H/I252Q/L264R,K024A/L075Q/D111A/G156W/V187T, K024A/L075Q/V077I/G156W/V187N,K024A/L075Q/V077I/V154I/V187N, K024A/L075R/V154I/G156W/V187Q,L075Q/D111A/V187N/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075Q/V154I/V187N/T189Q/L264R,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,L075R/G156W/V187T/T189Q/L264R, L075R/V077I/N094R/V154I/G156W,N011K/D027N/E056K/N233Q/P256T, N011K/D027N/E056K/S058M/P256T,N011K/G023K/D111A/G156W/L264R, N011K/G023K/E056K/L075Q/T189Q,P029E/N033D/E045F/L075D/T189D, P029E/N033D/K074S/L075D/T189D,P029E/N033D/N073R/L075D/T189D, Q004D/D027S/S058M/N233Q/P256T,Q004D/N011K/D027Q/E056K/N233Q, 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TABLE 8-11 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index >1 in the CS-61microswatch Assay using Tide ® full dose compared to Reference sequenceSEQ ID NO: 2 are shown below. L227M/L264R, A018K/L075D/T189D,A018K/P029E/T189D, A018K/V154I/G156W, D027S/N033D/T189D,G023K/V187N/L264R, G023Q/A049V/T189D, L075G/D130A/V187H,L075Q/V187N/L264R, L075R/D130A/V187T, L075R/V187N/L264R,P029E/N033D/E045F, V187N/T189Q/L264R, A018K/D027S/P029E/T189D,A018K/L075Q/V077I/N094R, A018K/N033D/L075D/T189D,D027S/N033D/L075D/T189D, D027S/P029E/L075D/T189D,D027S/P029E/N033D/E045F, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/D130A/V187T/L264R, P029E/N073R/L075D/T189D,A018K/D027S/P029E/N033D/L075D, A018K/G023Q/K024A/L075Q/G156W,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/P029E/N033D/N073R/L075D, D027E/D130A/G163P/L227M/L264R,D027E/D130A/G163P/N233Q/L264R, D027E/D137Q/G163P/N233Q/L264R,D027S/L075R/V154I/T189Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E045F/A049V/L075Q/V187T/T189D, E056K/L075R/D130A/V187T/L264R,G023K/D027S/E056K/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/E056K/L075R/V187N/L264R,G023K/E056K/L075R/V187T/L264R, G023K/L075Q/V077I/D130A/G156W,G023K/L075Q/V187H/I252Q/L264R, G023K/L075R/D130A/V187T/L264R,G023K/N094R/G156W/V187N/T189Q, K024A/L075R/V154I/G156W/V187Q,N011K/G023K/D111A/G156W/L264R, S058M/G163P/L227M/N233Q/L264R,A018K/G023K/L075Q/V077I/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/G156W,A018K/K024A/L075Q/N094R/V154I/G156W,A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/D137Q/G163P/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/N094R/D111A/G156W/V187T/L264R,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075Q/N094R/G156W/V187Q,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023K/L075Q/V077I/N094R/V154I/G156W,A018K/G023Q/L075R/V077I/N094R/G156W/V187N,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q,G023Q/D027S/E056K/L075R/D111A/V154I/G156W/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,A018K/G023Q/D027S/P029E/L075Q/V077I/R108K/H135F/V187H,G023K/K024A/L075Q/N094R/V154I/G156W/V187N/T189Q/L264R,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/H135F/G156W/V187T,A018K/G023Q/D027S/P029E/S058M/L075Q/V077I/R108K/H135F/V187H,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R

TABLE 8-12 TLL variants with Performance Index for expression ≥0.5compared to TLL SEQ ID NO: 1 and Performance Index ≥50% of the maximumPI value in the CS-61 microswatch Assay using Tide ® full dose comparedto Reference sequence SEQ ID NO: 2 are shown below. A018K/P029E/T189D,D027S/N033D/T189D, L075R/D130A/V187T, L075R/V187N/L264R,A018K/D027S/P029E/T189D, A018K/L075Q/V077I/N094R,A018K/N033D/L075D/T189D, D027S/P029E/N033D/L075D,D048Q/D130A/G163P/L264R, P029E/N073R/L075D/T189D,A018K/D027S/P029E/N033D/L075D, D027E/D130A/G163P/N233Q/L264R,D027E/D137Q/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,E056K/L075R/D130A/V187T/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/V187N/L264R, G023K/E056K/L075R/V187T/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187T/L264R,K024A/L075R/V154I/G156W/V187Q, A018K/K024A/L075Q/V077I/D130A/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D137Q/G163P/L227M/L264R,D027E/D048Q/D137Q/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D048Q/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/L075R/V187N/I252Q/L264R,G023K/K024A/L075Q/V154I/G156W/V187Q,A018K/D027S/P029E/N033D/N073R/L075D/T189D,A018K/G023K/K024A/L075R/N094R/D130A/V154I,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,Q004D/N011K/D027S/E056K/S058M/N233Q/P256T,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/F051T/E056K/L075Q/G091Q/V187H/I252Q

We claim:
 1. A lipolytic enzyme variant or an active fragment thereofcomprising an amino acid substitution at a position 130, wherein thelipolytic enzyme variant has at least 85% sequence identity to SEQ IDNO: 1 and the amino acid positions of the variant are numbered bycorrespondence with the amino acid sequence of Thermomyces lanuginosalipase set forth in SEQ ID NO: 1, and wherein the amino acidsubstitution is D130A.
 2. The lipolytic enzyme variant or activefragment thereof of claim 1, wherein the lipolytic enzyme variantfurther comprises amino acid substitutions selected from the groupconsisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73,75, 77, 90, 91, 94, 111, 137, 154, 156, 163, 187, 189, 227, 233, 252,256, and
 264. 3. The lipolytic enzyme variant or active fragment thereofof claim 2, wherein the variant or active fragment thereof furthercomprises amino acid modifications:G023/D027/F051/E056/S058/L075/G091/V187/I252/L264;G023/E056/L075/V187/L264; D027/D048/058/D137/G163/L227/N233/L264;A018/G023/K024/L075N077/N094/V154/G156/V187;A018/G023/K024/L075/N094/D111/V154/G156/V187/T189/L264; orN011/G023/D027/E056/L075/N094/D111/V154/G156/V187/T189/L264.
 4. Thelipolytic enzyme variant or active fragment thereof of claim 1, whereinthe variant or active fragment has lipolytic activity.
 5. The lipolyticenzyme variant or active fragment thereof of claim 1, wherein thevariant or active fragment has a performance index (pI) relative to theparent lipolytic enzyme for hydrolysis of p-nitrophenyl butyrate,p-nitrophenyl caprylate, or p-nitrophenyl palmitate that is greater than1.0.
 6. The lipolytic enzyme variant or active fragment thereof of claim5, wherein the performance index is measured using the p-nitrophenylbutyrate, p-nitrophenyl caprylate, or p-nitrophenyl palmitate assay ofExample 1, and, optionally, at a pH of
 8. 7. The lipolytic enzymevariant or active fragment thereof of any of claims 1-2, wherein thevariant or active fragment thereof comprises: (i) the amino acidmodifications selected from: D130A/L264R, D130A/V187N, L075Q/D130A,E056K/D130A/T189Q, L075G/D130A/V187H, L075Q/D111A/D130A,L075R/D130A/L264R, L075R/D130A/V187T, V077I/D130A/V154I,A018K/G023Q/L075R/D130A, A018K/L075Q/N094R/D130A,G023K/L075R/D130A/L264R, G023Q/L075Q/D130A/L264R,K024A/L075Q/D130A/V154I, L075Q/D130A/G156W/V187N,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/V0771/D130A/G156W,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075R/D130A/V1541,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, D027E/S058M/D130A/G163P/L264R,D027S/L075Q/D111A/D130A/V187N, D048Q/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,G023K/E056K/L075R/D130A/V187T, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/V0771/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023Q/K024A/L075R/D130A/V1541,G023Q/L075Q/V0771/D130A/G156W, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075R/D111A/D130A/V187N/T189Q,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,S058M/G163P/L227M/N233Q/L264R, A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/S058M/D130A/G163P/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075R/D130A/V154I/G156W/V187N/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023K/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,G023K/E056K/L075Q/D130A/V154I/N187N/T189Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,optionally wherein the variant or active fragment has a performanceindex (pI) relative to a reference lipolytic enzyme having the sequenceof SEQ ID NO:1 for hydrolysis of p-nitrophenyl butyrate that is greaterthan 1.0; (ii) the amino acid modifications selected from: D130A/L264R,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075R/D130A/L264R, L075R/D130A/V187T, A018K/G023Q/L075R/D130A,D048Q/D130A/G163P/L264R, D130A/G163P/L227M/L264R,G023K/L075R/D130A/L264R, K024A/L075Q/D130A/G156W,L075Q/D130A/G156W/V187N, L075Q/V077I/D130A/V187Q,L075R/D130A/V154I/L264R, A018K/G023Q/K024A/D130A/V154I,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,D027E/D130A/G163P/N233Q/L264R, D130A/D137Q/G163P/N233Q/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/V077I/D130A/G156W, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/V077I/D130A/G156W, GL075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, A018K/G023K/D111A/D130A/V154I/T189QA018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D048Q/S058M/D130A/G163P/N233Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023Q/K024A/L075Q/D130A/V154I/G156W,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R, optionally wherein thevariant or active fragment has a performance index (pI) relative to areference lipolytic enzyme having the sequence of SEQ ID NO:1 forhydrolysis of p-nitrophenyl caprylate that is greater than 1.0; and/or(iii) the amino acid modifications selected from: D130A/L264R,D130A/V187T, D111A/D130A/L264R, D130A/V187N/L264R, G023K/D130A/L264R,L075G/D130A/V187H, L075Q/D111A/D130A, L075R/D130A/L264R,L075R/D130A/V187T, A018K/G023Q/L075R/D130A, D027E/D130A/N233Q/L264R,D027S/D130A/I252Q/L264R, D048Q/D130A/G163P/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/L075Q/D130A/V187N, G023K/L075R/D130A/L264R,G023Q/L075Q/D130A/L264R, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, L075G/D130A/V187T/I252Q,L075Q/D130A/G156W/V187N, L075Q/V077I/D130A/V187Q,A018K/D130A/G156W/V187N/L264R, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075R/N094R/D130A, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/S058M/D130A/G163P/L264R,D027S/L075G/D130A/I252Q/L264R, D048Q/D130A/G163P/L227M/L264R,D130A/D137Q/G163P/L227M/L264R, D130A/D137Q/G163P/N233Q/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075Q/G091Q/D130A/L264R, G023K/E056K/D130A/V187N/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023Q/L075Q/D130A/I252Q/L264R,L075G/D130A/V187T/I252Q/L264R, L075R/D130A/V187T/T189Q/L264R,S058M/D130A/D137Q/G163P/L264R, S058M/D130A/G163P/L227M/L264R,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D130A/V187N/T189Q/L264R,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/L075Q/G091Q/V187T/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,A018K/G023K/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023K/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023K/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,optionally wherein the variant or active fragment has a performanceindex (pI) relative to a reference lipolytic enzyme having the sequenceof SEQ ID NO:1 for hydrolysis of D-nitrophenyl palmitate that is greaterthan 1.0.
 8. The lipolytic enzyme variant or active fragment thereof ofclaim 7, wherein the performance index for (i) hydrolysis ofp-nitrophenyl butyrate is measured using a p-nitrophenyl butyrate assay,(ii) hydrolysis of p-nitrophenyl caprylate is measured using ap-nitrophenyl caprylate assay, and/or (iii) hydrolysis of p-nitrophenylpalmitate is measured using a p-nitrophenyl palmitate assay.
 9. Thelipolytic enzyme variant or active fragment thereof of claim 8, whereinthe performance index is measured at a pH of
 8. 10. The lipolytic enzymevariant or active fragment thereof of claim 1, wherein either: (a) thevariant comprises amino acid modifications selected from: D130A/L264R,D130A/T189Q, D130A/V187N, D130A/V187T, G023K/D130A,L075Q/D130A,D111A/D130A/L264R, D130A/T189Q/L264R, D130A/V154I/G156W,D130A/V187N/L264R, D130A/V187T/L264R, E056K/D130A/L264R,E056K/D130A/T189Q, G023K/D130A/L264R, G023K/D130A/V187T,G023Q/D130A/G156W, K024A/D130A/V154I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/D130A/V187T, L075R/D130A/L264R,L075R/D130A/V187T, N094R/D130A/V187T, V077I/D130A/V154I,A018K/D130A/G156W/V187T, A018K/G023Q/L075R/D130A,A018K/K024A/D130A/G156W, A018K/L075Q/N094R/D130A,D027E/D130A/N233Q/L264R, D048Q/D130A/G163P/L264R,D111A/D130A/V154I/G156W, D111A/D130A/V154I/L264R,D130A/G163P/L227M/L264R, D130A/G163P/N233Q/L264R,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,F051T/D130A/I252Q/L264R, G023K/D111A/D130A/L264R,G023K/D130A/G156W/T189Q, G023K/D130A/V187N/L264R,G023K/D130A/V187T/L264R, G023K/D130A/V187T/T189Q,G023K/E056K/D130A/L264R, G023K/L075Q/D130A/V187N,G023K/L075R/D130A/L264R, G023Q/K024A/D130A/G156W,G023Q/L075Q/D130A/L264R, K024A/D130A/V154I/N187T,K024A/L075Q/D130A/G156W, K024A/L075Q/D130A/V154I,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/V077I/D130A/V187Q,L075R/D130A/V154I/L264R, L075R/D130A/V187T/L264R,A018K/D130A/G156W/V187N/L264R, A018K/D130A/G156W/V187T/L264R,A018K/G023K/D130A/V154I/G156W, A018K/G023K/K024A/D130A/G156W,A018K/G023K/K024A/D130A/V154I, A018K/G023K/L075Q/D130A/V154I,A018K/G023K/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/D130A/V187Q, A018K/K024A/L075Q/D130A/L264R,A018K/K024A/L075R/D130A/V154I, A018K/K024A/L075R/N094R/D130A,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, D027E/D130A/D137Q/G163P/L264R,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027Q/F051T/L075Q/D130A/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075Q/D111A/D130A/V187N,D048Q/D130A/D137Q/G163P/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/G163P/L227M/N233Q/L264R,D130A/V154I/G156W/V187N/T189Q, E056K/D130A/V187H/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, E056K/N094R/G156W/V187N/L264R,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/K024A/D111A/D130A/V187T,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/D130A/G156W/L264R, G023K/L075Q/D130A/V154I/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023Q/K024A/L075R/D130A/V154I,G023Q/K024A/L075R/V077I/D130A, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,G091Q/D130A/V187H/I252Q/L264R, L075G/D130A/V187T/I252Q/L264R,L075Q/D111A/D130A/V187T/T189Q, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075Q/V077I/D130A/G156W/V187N,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, V077I/D130A/V154I/G156W/V187N,A018K/G023K/D111A/D130A/V154I/T189Q,A018K/G023K/K024A/L075R/D130A/V187N,A018K/G023K/K024A/N094R/D130A/V154I,A018K/G023K/L075Q/D130A/G156W/V187N,A018K/G023K/L075R/N094R/D130A/V187N,A018K/G023K/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,F051T/L075R/D130A/V187N/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075G/G091Q/D130A/I252Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D111A/D130A/V154I/V187N/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/N094R/D130A/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023K/K024A/L075Q/D130A/G156W/V187N,A018K/G023K/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/K024A/N094R/D130A/G156W/V187T/L264R,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/D130A/G156W/V187N/T189Q/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/V187N/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,A018K/G023K/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023K/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023K/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023G/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023G/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023G/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,A018K/G023G/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023G/K024A/L075Q/N094R/D130A/V187T/T189Q/L264K,A018K/G023G/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023G/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,N011K/A018K/G023G/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023G/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,optionally wherein the variant or active fragment has a performanceindex (pI) relative to a reference lipolytic enzyme having the sequenceof SEQ ID NO: 1 for thermostability that is greater than 1.0; or, (b)the variant comprises amino acid modifications selected from:D130A/T189Q, D130A/V187N, D130A/V187T, G023K/D130A, L075Q/D130A,D111A/D130A/L264R, D130A/V154I/G156W, E056K/D130A/L264R,G023K/D130A/V187T, G023Q/D130A/G156W, K024A/D130A/V154I,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075R/D130A/L264R, L075R/D130A/V187T, N094R/D130A/V187T,V077I/D130A/V154I, A018K/D130A/G156W/V187T, A018K/G023Q/L075R/D130A,A018K/K024A/D130A/G156W, A018K/L075Q/N094R/D130A,D111A/D130A/V154I/G156W, D111A/D130A/V154I/L264R,D111A/D130A/V187T/L264R, E056K/D130A/V187T/L264R,F051T/D130A/I252Q/L264R, G023K/D111A/D130A/L264R,G023K/D130A/G156W/T189Q, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/L075Q/D130A/V187N,G023Q/K024A/D130A/G156W, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, K024A/L075Q/D130A/V154I,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/V077I/D130A/V187Q,L075R/D130A/V154I/L264R, A018K/G023G/D130A/V154I/G156W,A018K/G023G/K024A/D130A/G156W, A018K/G023G/K024A/D130A/V154I,A018K/G023G/L075Q/D130A/V154I, A018K/G023G/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/D130A/V187Q,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,D130A/G163P/L227M/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/D130A/V187H/I252Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/K024A/D111A/D130A/V187T, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/D130A/V154I/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023Q/K024A/L075Q/D130A/G156W,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/V077I/D130A,G023Q/K024A/V077I/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,L075Q/D111A/D130A/V187T/T189Q, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075Q/V077I/D130A/G156W/V187N,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/G023G/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/D130A/V187N,A018K/G023G/K024A/N094R/D130A/V154I,A018K/G023G/L075Q/D130A/G156W/V187N,A018K/G023G/L075R/N094R/D130A/V187N,A018K/G023G/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/G091Q/D130A/V187N/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D111A/D130A/V154I/V187N/T189Q,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023G/K024A/L075Q/D130A/G156W/V187N,A018K/G023G/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,A018K/G023G/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023G/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023G/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023G/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023G/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023G/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,A018K/G023G/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023G/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R, optionallywherein the variant or active fragment has a performance index (pI) fordetergent stability which is greater than 1.0; or, (c) the variantcomprises amino acid modifications selected from: D130A/T189Q,D130A/V187N, D130A/V187T, G023K/D130A, L075Q/D130A, D111A/D130A/L264R,D130A/V154I/G156W, E056K/D130A/L264R, G023K/D130A/V187T,G023Q/D130A/G156W, K024A/D130A/V154I, L075G/D130A/V187H,L075Q/D111A/D130A, L075Q/D130A/V187T, L075R/D130A/L264R,L075R/D130A/V187T, N094R/D130A/V187T, V077I/D130A/V154I,A018K/D130A/G156W/V187T, A018K/G023Q/L075R/D130A,A018K/K024A/D130A/G156W, A018K/L075Q/N094R/D130A,D111A/D130A/V154I/G156W, D111A/D130A/V154I/L264R,D111A/D130A/V187T/L264R, E056K/D130A/V187T/L264R,F051T/D130A/I252Q/L264R, G023K/D111A/D130A/L264R,G023K/D130A/G156W/T189Q, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/L075Q/D130A/V187N,G023Q/K024A/D130A/G156W, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, K024A/L075Q/D130A/V154I,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/V077I/D130A/V187Q,L075R/D130A/V154I/L264R, A018K/G023G/D130A/V154I/G156W,A018K/G023G/K024A/D130A/G156W, A018K/G023G/K024A/D130A/V154I,A018K/G023G/L075Q/D130A/V154I, A018K/G023G/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/K024A/D130A/V187Q,A018K/K024A/L075R/D130A/V154I, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,D130A/G163P/L227M/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,E056K/D130A/V187H/I252Q/L264R, E056K/L075R/D130A/V187T/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/K024A/D111A/D130A/V187T, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/D130A/V154I/T189Q, G023K/L075Q/V077I/D130A/G156W,G023K/L075R/D130A/V187N/L264R, G023Q/K024A/L075Q/D130A/G156W,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/L075R/V077I/D130A,G023Q/K024A/V077I/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,L075Q/D111A/D130A/V187T/T189Q, L075Q/D130A/V187T/T189Q/L264R,L075Q/N094R/D130A/G156W/V187T, L075Q/V077I/D130A/G156W/V187N,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,V077I/D130A/V154I/G156W/V187N, A018K/G023G/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/D130A/V187N,A018K/G023G/K024A/N094R/D130A/V154I,A018K/G023G/L075Q/D130A/G156W/V187N,A018K/G023G/L075R/N094R/D130A/V187N,A018K/G023G/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/G091Q/D130A/V187N/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D111A/D130A/V154I/V187N/T189Q,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023G/K024A/L075Q/D130A/G156W/V187N,A018K/G023G/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,A018K/G023G/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023G/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023G/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023G/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023G/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023G/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/E045F/L075Q/V077I/G156W/V187T/T189D,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/N187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,A018K/G023G/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023G/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R, optionallywherein the variant or active fragment has a performance index (pI) forLAS stability which is greater than 1.0; or, (d) the variant comprisesamino acid modifications selected from: D130A/L264R, D130A/V187N,D130A/V187T, D111A/D130A/L264R, D130A/V187N/L264R, D130A/V187T/L264R,G023K/D130A/L264R, G023K/D130A/V187T, L075R/D130A/L264R,L075R/D130A/V187T, A018K/G023Q/L075R/D130A, A018K/K024A/D130A/G156W,D027E/D130A/N233Q/L264R, D027S/D130A/I252Q/L264R,D048Q/D130A/G163P/L264R, D048Q/G163P/N233Q/L264R,D111A/D130A/V154I/G156W, D111A/D130A/V187T/L264R,E056K/D130A/V187N/L264R, E056K/D130A/V187T/L264R,G023K/D130A/V187N/L264R, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/L075Q/D130A/V187N,G023Q/L075Q/D130A/L264R, K024A/D130A/V154I/V187T,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/V077I/D130A/V187Q, L075R/D130A/V154I/L264R,A018K/D130A/G156W/V187N/L264R, A018K/G023G/K024A/D130A/G156W,A018K/G023G/K024A/D130A/V154I, A018K/G023G/V077I/D130A/V187N,A018K/G023Q/K024A/D130A/V187Q, A018K/K024A/L075R/N094R/D130A,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027S/L075G/D130A/I252Q/L264R,D048Q/D130A/G163P/L227M/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, D130A/V154I/G156W/V187N/T189Q,F051T/D130A/V187T/I252Q/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075G/G091Q/D130A/L264R, F051T/L075Q/G091Q/D130A/L264R,G023K/E056K/D130A/V187N/L264R, G023K/E056K/D130A/V187T/L264R,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187N/L264R,G023K/L075R/D130A/V187T/L264R, G023Q/L075Q/D130A/I252Q/L264R,G091Q/D130A/V187H/I252Q/L264R, L075G/D130A/V187T/I252Q/L264R,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,S058M/D130A/G163P/L227M/L264R, A018K/G023G/K024A/L075R/D130A/V187N,A018K/G023G/L075Q/D130A/G156W/V187N,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137H/G163P/L264R,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/L227M/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/E056K/D130A/V187N/I252Q/L264R,F051T/L075R/D130A/V187N/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187T/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/L075Q/D130A/I252Q/L264R,G023K/L075Q/D111A/D130A/V154I/V187N,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/I252Q/L264R,L075Q/D111A/D130A/V154I/V187N/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/D111A/D130A/V154I/G156W/V187T/T189Q,A018K/G023G/K024A/L075Q/D130A/G156W/V187N,A018K/G023Q/K024A/L075R/D130A/V154I/V187N,A018K/K024A/D130A/V154I/G156W/V187T/L264R,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/N094R/D130A/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/N233Q/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/G163P/L227M/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187T/I252Q/L264R,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/F051T/E056K/D130A/I252Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/S058M/G091Q/D130A/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V154I/G156W/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,A018K/G023G/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023Q/D027Q/F051T/L075Q/G091Q/D130A/I252Q/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/S058M/L075R/G091Q/D130A/V187H/I252Q/L264R,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,A018K/G023G/K024A/L075Q/N094R/D130A/V187T/T189Q/L264K,A018K/G023G/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023G/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023K/L075Q/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/F051T/S058M/L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/V187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,N011K/A018K/G023G/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R, optionallywherein the variant or active fragment has a performance index (pI) forfabric adhesion which is greater than 1.0, or, (e) the variant comprisesamino acid modifications selected from: D130A/T189Q, G023K/D130A,L075Q/D130A, D130A/T189Q/L264R, D130A/V154I/G156W, E056K/D130A/L264R,E056K/D130A/T189Q, G023Q/D130A/G156W, K024A/D130A/V154I,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,N094R/D130A/V187T, V077I/D130A/V154I, A018K/D130A/G156W/V187T,A018K/L075Q/N094R/D130A, D111A/D130A/V154I/L264R,D130A/G163P/L227M/L264R, D130A/G163P/N233Q/L264R,F051T/D130A/I252Q/L264R, G023K/D111A/D130A/L264R,G023K/D130A/G156W/T189Q, G023K/E056K/D130A/L264R,G023K/E056K/D130A/V187N, G023K/L075R/D130A/L264R,G023Q/K024A/D130A/G156W, K024A/L075Q/D130A/G156W,K024A/L075Q/D130A/V154I, L075Q/D130A/V154I/G156W,L075R/D130A/V187T/L264R, A018K/D130A/G156W/V187T/L264R,A018K/G023G/D130A/V154I/G156W, A018K/G023G/L075Q/D130A/V154I,A018K/G023Q/K024A/D130A/V154I, A018K/G023Q/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075R/D130A/V154I,A018K/L075Q/N094R/D130A/V187N, D027E/D130A/D137Q/G163P/L264R,D027Q/F051T/L075Q/D130A/L264R, D027S/L075Q/D111A/D130A/V187N,D048Q/D130A/D137Q/G163P/L264R, D048Q/S058M/D130A/N233Q/L264R,D130A/G163P/L227M/N233Q/L264R, E056K/D130A/V187H/I252Q/L264R,E056K/L075R/D130A/V187N/L264R, E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/K024A/D111A/D130A/V187T,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D130A/V154I/T189Q,G023Q/K024A/L075Q/D130A/G156W, G023Q/K024A/L075R/D130A/V154I,G023Q/K024A/L075R/V077I/D130A, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/V077I/D130A/G156W, L075Q/D111A/D130A/V187T/T189Q,L075Q/D130A/V187T/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075R/D111A/D130A/V187N/T189Q,S058M/D130A/D137Q/G163P/L264R, V077I/D130A/V154I/G156W/V187N,A018K/G023G/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/N094R/D130A/V154I,A018K/G023G/L075R/N094R/D130A/V187N,A018K/G023G/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/K024A/V077I/N094R/D130A/G156W,D027E/D048Q/S058M/D130A/G163P/L264R,D027E/D048Q/S058M/D130A/L227M/N233Q,D027E/D130A/D137Q/G163P/L227M/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027S/G091Q/D130A/V187N/I252Q/L264R,D048Q/D130A/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/E056K/D111A/D130A/V187N/T189Q,G023Q/E056K/L075Q/D130A/V154I/L264R,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/N094R/D130A/L264R,A018K/G023G/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,A018K/L075Q/D130A/G156W/V187N/T189Q/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/L075Q/D111A/D130A/V154I/G156W/L264R,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023K/L075Q/N094R/D130A/V154I/V187N/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D111A/D130A/V154I/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,A018K/G023G/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023G/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023G/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023G/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023G/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/E056K/L075Q/D130A/V154I/V187N/T189Q/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/D111A/D130A/V154I/G156W/V187N/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/D027S/L075Q/D111A/D130A/V154I/T189Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/N187N/L264R,A018K/G023G/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075Q/N094R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/G023Q/D027S/E056K/L075R/D130A/V154I/G156W/T189Q/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023G/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,optionally wherein the variant or active fragment has a performanceindex (pI) for fabric adhesion which is less than 1.0, or, (f) thevariant comprises amino acid modifications selected from: D130A/L264R,D130A/V187T, G023K/D130A, L075Q/D130A, D111A/D130A/L264R,E056K/D130A/T189Q L075G/D130A/V187H L075Q/D111A/D130A,L075Q/D130A/V187T, L075R/D130A/L264R, L075R/D130A/V187T,V077I/D130A/V154I, A018K/D130A/G156W/V187T, A018K/G023Q/L075R/D130A,D027E/D130A/N233Q/L264R, D027S/D130A/I252Q/L264R,D048Q/D130A/G163P/L264R, D111A/D130A/V154I/L264R,D130A/G163P/L227M/L264R, D130A/G163P/N233Q/L264R,E056K/D130A/V187N/L264R, F051T/D130A/I252Q/L264R,G023K/D130A/G156W/T189Q, G023K/D130A/V187T/L264R,G023K/D130A/V187T/T189Q, G023K/L075Q/D130A/V187N,G023K/L075R/D130A/L264R, G023Q/L075Q/D130A/L264R,K024A/D130A/V154I/V187T, K024A/L075Q/D130A/G156W,L075G/D130A/V187T/I252Q, L075Q/D130A/G156W/V187N,L075Q/D130A/V154I/G156W, L075Q/V077I/D130A/V187Q,L075R/D130A/V154I/L264R, L075R/D130A/V187T/L264R,A018K/D130A/G156W/V187N/L264R, A018K/G023G/D130A/V154I/G156W,A018K/G023G/K024A/D130A/G156W, A018K/G023G/L075Q/D130A/V154I,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075R/N094R/D130A,A018K/K024A/N094R/D130A/V187N, A018K/L075Q/D111A/D130A/V187T,A018K/L075Q/N094R/D130A/V187N, D027E/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/G163P/L264R, D027S/L075G/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L264R, D048Q/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/N233Q/L264R, D130A/D137Q/G163P/L227M/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/D130A/V187H/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, F051T/L075G/D130A/I252Q/L264R,F051T/L075Q/G091Q/D130A/L264R, G023K/E056K/D130A/V187T/L264R,G023K/E056K/L075R/D130A/V187T, G023K/K024A/L075R/D130A/G156W,G023K/L075Q/D111A/D130A/V187T, G023K/L075Q/D130A/G156W/L264R,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187T/L264R,G023Q/K024A/L075R/D130A/V154I, G023Q/K024A/V077I/D130A/V154I,G023Q/L075Q/D130A/I252Q/L264R, G023Q/L075Q/V077I/D130A/G156W,G091Q/D130A/V187H/I252Q/L264R, L075G/D130A/V187T/I252Q/L264R,L075Q/D111A/D130A/V187T/T189Q, L075Q/N094R/D130A/G156W/V187T,L075R/D111A/D130A/V187N/T189Q, L075R/D130A/V154I/T189Q/L264R,L075R/D130A/V187T/T189Q/L264R, S058M/D130A/D137Q/G163P/L264R,S058M/D130A/G163P/L227M/L264R, A018K/G023G/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/D130A/V187N,A018K/G023G/K024A/N094R/D130A/V154I,A018K/G023G/L075Q/D130A/G156W/V187N,A018K/G023G/L075R/N094R/D130A/V187N,A018K/G023G/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D048Q/D130A/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/D137Q/G163P/N233Q/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/G163P/L227M/L264R,D027E/S058M/D130A/G163P/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/G091Q/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/L075Q/D130A/V187T/I252Q/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/F051T/L075Q/D130A/I252Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L264R,D048Q/S058M/D130A/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/D130A/V154I/G156W/V187T/L264R,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/F051T/G091Q/D130A/V187H/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/V187N,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/L075G/D130A/V187H/L264R,G023Q/D027S/L075Q/D130A/V187T/I252Q,G023Q/F051T/D130A/V187T/I252Q/L264R,G023Q/K024A/D130A/V154I/G156W/V187Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/V077I/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/N094R/D130A/V154I/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D111A/D130A/V154I/V187N/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075Q/G091Q/D130A/V187H/I252Q/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/N094R/D130A/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/G023G/K024A/L075Q/D130A/G156W/V187N,A018K/G023G/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/D130A/V154I/N187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,D027E/D048Q/S058M/D130A/D137Q/G163P/L264R,D027E/D048Q/S058M/D130A/G163P/L227M/L264R,D027E/D048Q/S058M/D130A/G163P/N233Q/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D027Q/L075G/G091Q/D130A/V187H/I252Q/L264R,D027S/F051T/S058M/L075R/D130A/I252Q/L264R,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/L264R,D048Q/S058M/D130A/G163P/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,F051T/L075G/G091Q/D130A/V187N/I252Q/L264R,F051T/L075R/G091Q/D130A/V187T/I252Q/L264R,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/D027S/F051T/S058M/L075Q/D130A/V187N,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/F051T/L075Q/G091Q/D130A/V187H/I252Q,G023K/K024A/L075Q/D111A/D130A/T189Q/L264R,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D130I/V154I/G156W/T189Q,G023Q/E056K/L075R/D111A/D130A/V187N/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,G023Q/S058M/L075G/G091Q/D130A/I252Q/L264R,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,A018K/G023G/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023G/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023G/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023G/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023G/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,F051T/E056K/L075G/G091Q/D130A/V187H/I252Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027Q/F051T/S058M/L075R/D130A/V187T/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/D027S/S058M/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187H/I252Q/L264R,G023Q/F051T/L075Q/G091Q/D130A/V187T/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,N011K/G023Q/L075Q/D111A/D130A/V154I/V187N/L264R,A018K/G023G/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023G/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027Q/F051T/S058M/L075G/G091Q/D130A/V187T/I252Q/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,N011K/G023K/L075R/D111A/D130A/V154I/N187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023G/K024A/L075R/V077I/D130A/V154I/V187T/T189Q,N011K/G023Q/E056K/L075R/D111A/D130A/V154I/G156W/V187N/L264R,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023G/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,optionally wherein the variant or active fragment has a performanceindex (pI) for cleaning performance which is greater than 1.0; or, (g)the variant comprises amino acid modifications selected from:D130A/L264R, D130A/V187N, G023K/D130A, L075Q/D130A, D111A/D130A/L264R,E056K/D130A/T189Q, G023K/D130A/L264R, G023K/D130A/V187T,L075G/D130A/V187H, L075Q/D111A/D130A, L075Q/D130A/V187T,L075R/D130A/L264R, L075R/D130A/V187T, V077I/D130A/V154I,A018K/G023Q/L075R/D130A, D048Q/D130A/G163P/L264R,D111A/D130A/V154I/L264R, D130A/G163P/L227M/L264R,D130A/G163P/N233Q/L264R, E056K/D130A/V187N/L264R,E056K/D130A/V187T/L264R, G023K/D130A/V187T/L264R,G023K/E056K/D130A/L264R, G023K/L075Q/D130A/V187N,G023K/L075R/D130A/L264R, K024A/D130A/V154I/V187T,K024A/L075Q/D130A/G156W, L075G/D130A/V187T/I252Q,L075Q/D130A/G156W/V187N, L075Q/D130A/V154I/G156W,L075Q/V077I/D130A/V187Q, L075R/D130A/V154I/L264R,A018K/D130A/G156W/V187N/L264R, A018K/G023G/D130A/V154I/G156W,A018K/G023G/K024A/D130A/G156W, A018K/G023G/L075Q/D130A/V154I,A018K/G023G/V077I/D130A/V187N, A018K/G023Q/K024A/D130A/V154I,A018K/G023Q/K024A/D130A/V187Q, A018K/G023Q/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/L264R, A018K/K024A/L075R/D130A/V154I,A018K/K024A/L075R/N094R/D130A, A018K/K024A/N094R/D130A/V187N,A018K/L075Q/D111A/D130A/V187T, A018K/L075Q/N094R/D130A/V187N,D027E/D130A/G163P/L227M/L264R, D027E/D130A/G163P/N233Q/L264R,D027S/L075G/D130A/I252Q/L264R, D027S/L075Q/D111A/D130A/V187N,D048Q/D130A/D137Q/G163P/L264R, D048Q/S058M/D130A/N233Q/L264R,D130A/D137Q/G163P/N233Q/L264R, E056K/D130A/V187H/I252Q/L264R,E056K/L075R/D130A/V187T/L264R, F051T/L075Q/G091Q/D130A/L264R,G023K/E056K/D130A/V187T/L264R, G023K/E056K/L075R/D130A/V187T,G023K/K024A/L075R/D130A/G156W, G023K/L075Q/D111A/D130A/V187T,G023K/L075Q/D130A/G156W/L264R, G023K/L075Q/D130A/V154I/T189Q,G023K/L075Q/V077I/D130A/G156W, G023K/L075R/D130A/V187T/L264R,G023Q/K024A/L075Q/D130A/G156W, G023Q/K024A/L075R/D130A/V154I,G023Q/K024A/L075R/G156W/V187Q, G023Q/K024A/L075R/V077I/D130A,G023Q/K024A/V077I/D130A/V154I, G023Q/L075Q/V077I/D130A/G156W,G091Q/D130A/V187H/I252Q/L264R, L075Q/D111A/D130A/V187T/T189Q,L075Q/D111A/V187N/T189Q/L264R, L075Q/N094R/D130A/G156W/V187T,L075Q/V077I/D130A/G156W/V187N, L075R/D111A/D130A/V187N/T189Q,L075R/D130A/V154I/T189Q/L264R, L075R/D130A/V187T/T189Q/L264R,S058M/D130A/D137Q/G163P/L264R, A018K/G023G/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/D130A/V187N,A018K/G023G/K024A/N094R/D130A/V154I,A018K/G023G/L075Q/D130A/G156W/V187N,A018K/G023G/L075R/N094R/D130A/V187N,A018K/G023G/L075R/V077I/D130A/V154I,A018K/G023Q/L075Q/V077I/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V187N,A018K/G023Q/N094R/D130A/G156W/V187Q,A018K/K024A/L075Q/V077I/D130A/V187N,A018K/K024A/L075R/D130A/V187N/T189Q,A018K/K024A/V077I/N094R/D130A/G156W,A018K/L075Q/D130A/V154I/T189Q/L264R,A018K/L075Q/D130A/V187N/T189Q/L264R,A018K/L075R/D130A/V154I/G156W/V187N,A018K/N094R/D111A/D130A/V154I/V187N,D027E/D048Q/D130A/D137Q/G163P/L264R,D027E/D130A/D137Q/G163P/L227M/L264R,D027E/D130A/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/L227M/N233Q/L264R,D027Q/E056K/S058M/D130A/I252Q/L264R,D027Q/F051T/L075G/D130A/I252Q/L264R,D027Q/F051T/L075Q/D130A/V187H/L264R,D027Q/S058M/L075R/D130A/V187T/I252Q,D027S/G091Q/D130A/V187N/I252Q/L264R,D048Q/S058M/D130A/G163P/N233Q/L264R,D111A/D130A/V154I/G156W/V187N/L264R,F051T/L075G/G091Q/D130A/I252Q/L264R,G023K/D027S/L075R/D130A/V187T/I252Q,G023K/E056K/L075R/D130A/T189Q/L264R,G023K/E056K/L075R/D130A/V187N/L264R,G023K/F051T/D130A/V187N/I252Q/L264R,G023K/K024A/L075Q/D111A/D130A/T189Q,G023K/K024A/L075Q/N094R/D130A/G156W,G023K/K024A/L075R/D130A/V154I/N187N,G023K/L075G/D130A/V187T/I252Q/L264R,G023K/L075Q/D111A/D130A/V154I/V187N,G023K/L075Q/V077I/D130A/G156W/V187Q,G023Q/F051T/L075Q/D130A/V187H/I252Q,G023Q/K024A/L075Q/D130A/V154I/G156W,G023Q/K024A/L075Q/D130A/V154I/V187N,G023Q/K024A/V077I/D130A/V154I/G156W,G023Q/L075Q/V077I/N094R/D130A/V187Q,K024A/D111A/D130A/V187T/T189Q/L264R,K024A/L075Q/D130A/G156W/T189Q/L264R,K024A/L075Q/V077I/N094R/D130A/V187N,L075Q/D111A/D130A/V154I/G156W/T189Q,L075Q/D111A/D130A/V154I/V187N/T189Q,L075Q/D130A/V154I/G156W/V187N/L264R,L075R/D130A/V154I/G156W/V187N/L264R,N011K/E056K/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/D130A/V187N/T189Q,N011K/G023Q/L075Q/N094R/D130A/L264R,S058M/D130A/D137Q/G163P/L227M/L264R,S058M/D130A/D137Q/G163P/N233Q/L264R,S058M/D130A/G163P/L227M/N233Q/L264R,A018K/G023G/K024A/L075Q/D130A/G156W/V187N,A018K/G023G/K024A/L075R/N094R/D130A/V154I,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W,A018K/G023Q/K024A/L075R/D130A/V154I/N187N,A018K/G023Q/K024A/L075R/V077I/D130A/G156W,A018K/K024A/L075Q/D130A/G156W/V187T/T189Q,A018K/K024A/L075Q/D130A/V154I/G156W/V187T,A018K/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/L075Q/D111A/D130A/V154I/G156W/L264R,D027E/D130A/D137Q/G163P/L227M/N233Q/L264R,D027E/S058M/D130A/D137Q/G163P/L227M/L264R,D027E/S058M/D130A/D137Q/G163P/N233Q/L264R,D027Q/F051T/L075G/D130A/V187H/I252Q/L264R,D027Q/F051T/L075Q/G091Q/D130A/V187T/I252Q,D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/L227M/N233Q/L264R,E056K/L075Q/D111A/D130A/G156W/V187N/T189Q,G023K/D027Q/F051T/L075G/D130A/V187H/L264R,G023K/F051T/E056K/L075R/D130A/V187T/I252Q,G023K/K024A/L075Q/D130A/V187T/T189Q/L264R,G023K/K024A/L075Q/V077I/D130A/G156W/V187Q,G023K/L075Q/D130A/G156W/V187N/T189Q/L264R,G023Q/D027S/S058M/L075Q/D130A/I252Q/L264R,G023Q/E056K/L075Q/D130A/V154I/G156W/T189Q,G023Q/F051T/G091Q/D130A/V187H/I252Q/L264R,G023Q/K024A/L075Q/V077I/D130A/G156W/V187N,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W,G023Q/L075Q/D111A/D130A/V154I/G156W/L264R,G023Q/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/D130A/V154I/V187N/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/G156W/V187N,G023Q/L075Q/V077I/N094R/D130A/G156W/V187Q,K024A/L075Q/D111A/D130A/V187N/T189Q/L264R,K024A/L075R/D130A/V154I/G156W/T189Q/L264R,L075Q/D111A/D130A/G156W/V187N/T189Q/L264R,N011K/D027S/D111A/D130A/V154I/V187N/T189Q,N011K/E056K/L075Q/D130A/V154I/V187N/T189Q,N011K/G023K/L075Q/D111A/D130A/V154I/V187N,A018K/G023G/K024A/L075Q/D130Y/V154I/V187N/T189Q,A018K/G023G/K024A/L075Q/N094R/D130A/G156W/V187Q,A018K/G023G/K024A/L075Q/V077I/D130A/V154I/G156W,A018K/G023G/K024A/L075Q/V077I/N094R/D130A/G156W,A018K/G023G/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/G023G/L075R/V077I/N094R/D130A/G156W/V187Q,A018K/G023Q/K024A/L075Q/V077I/D130A/G156W/V187Q,A018K/G023Q/K024A/L075R/D130A/V154I/G156W/V187N,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W,A018K/G023Q/K024A/L075R/V077I/N094R/D130A/G156W,A018K/G023Q/L075Q/V077I/D130A/V154I/G156W/V187N,A018K/K024A/L075Q/V077I/N094R/D130A/V154I/G156W,A018K/K024A/L075R/D130A/V154I/G156W/V187T/L264R,D027E/D048Q/D130A/D137Q/G163P/L227M/N233Q/L264R,D048Q/S058M/D130A/D137Q/G163P/L227M/N233Q/L264R,G023K/D027S/E056K/L075G/D130A/V187T/I252Q/L264R,G023K/D027S/F051T/E056K/L075R/D130A/V187T/L264R,G023K/F051T/E056K/L075R/G091Q/D130A/V187T/L264R,G023K/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,G023Q/D027S/F051T/S058M/G091Q/D130A/V187N/I252Q,G023Q/D027S/L075Q/D130A/V154I/G156W/T189Q/L264R,G023Q/K024A/L075Q/V077I/D130A/V154I/G156W/V187N,G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/V187N,G023Q/L075Q/N094R/D111A/D130A/G156W/T189Q/L264R,G023Q/L075Q/N094R/D130A/V154I/G156W/T189Q/L264R,G023Q/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,K024A/D111A/D130A/V154I/G156W/V187T/T189Q/L264R,N011K/G023K/E056K/L075Q/D130A/V154I/T189Q/L264R,A018K/G023G/K024A/L075Q/N094R/D111A/D130A/V154I/T189Q,A018K/G023G/K024A/L075R/V077I/D130A/V154I/G156W/V187N,A018K/G023G/K024A/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075R/N094R/D130A/V154I/G156W/V187N,D027S/E056K/L075Q/N094R/D111A/D130A/V154I/G156W/L264R,D027S/E056K/L075Q/N094R/D111A/D130A/V187N/T189Q/L264R,G023K/D027S/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/E056K/L075Q/D111A/D130A/V154I/G156W/T189Q/L264R,G023K/K024A/N094R/D130A/V154I/G156W/V187T/T189Q/L264R,G023Q/D027S/L075Q/N094R/D130A/V154I/G156W/V187N/L264R,N011K/G023K/L075R/D111A/D130A/V154I/N187N/T189Q/L264R,N011K/G023K/L075R/V077I/D130A/V154I/G156W/V187T/T189Q,A018K/G023Q/K024A/L075Q/V077I/N094R/D130A/V154I/G156W/V187Q,G023K/D027S/F051T/E056K/S058M/L075G/G091Q/D130A/I252Q/L264R,G023Q/D027S/F051T/E056K/L075R/G091Q/D130A/V187H/I252Q/L264R,G023Q/D027S/F051T/E056K/S058M/L075R/G091Q/D130A/V187H/I252Q,G023Q/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,N011K/A018K/G023G/K024A/L075R/V077I/D130A/V154I/N187T/T189Q,N011K/G023Q/L075Q/N094R/D111A/D130A/V154I/G156W/V187N/L264R,A018K/G023G/L075R/N094R/D111A/D130A/V154I/G156W/V187N/T189Q/L264R,optionally wherein the variant or active fragment has a performanceindex (pI) for cleaning performance which is greater than 1.0.
 11. Acomposition comprising the lipolytic enzyme variant of claim
 1. 12. Thecomposition of claim 11, wherein said composition is a granular, powder,solid, bar, liquid, tablet, gel, unit dose, or paste composition. 13.The composition of claim 11, wherein said composition is a laundrydetergent composition, a dish detergent composition, or a hard surfacecleaning composition.
 14. The composition of claim 11, furthercomprising at least one bleaching agent and/or at least one additionalenzyme selected from the group consisting of protease, hemicellulase,cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme,xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase,pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase,phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase,pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase,chondroitinase, laccase, and amylase.
 15. A method of cleaning,comprising contacting a surface or an item with the composition of claim11.
 16. The method of claim 15, wherein said item is dishware or fabric.